Homotrimeric extended obg3 globular head and uses thereof

ABSTRACT

The present invention relates to the field of obesity research. Obesity is a public health problem that is serious and widespread. A compound, homotrimer of OBG3 polypeptide fragment comprising globular domain and all or part of OBG3 collagen-like region, has been identified that has utility for reducing body mass, for maintaining weight loss, and for treating obesity-related diseases and disorders. These obesity-related diseases and disorders include hyperlipidemias, atherosclerosis, diabetes, and hypertension.

FIELD OF THE INVENTION

The present invention relates to the field of metabolic research, inparticular the discovery of compounds effective for treating obesity andobesity-related diseases and disorders. The obesity-related diseases ordisorders envisioned to be treated by the methods of the inventioninclude, but are not limited to, hyperlipidemia, atherosclerosis,diabetes, and hypertension.

BACKGROUND OF THE INVENTION

The following discussion is intended to facilitate the understanding ofthe invention, but is not intended nor admitted to be prior art to theinvention.

Obesity is a public health problem that is serious, widespread, andincreasing. In the United States, 20 percent of the population is obese;in Europe, a slightly lower percentage is obese [Friedman (2000) Nature404:632-634]. Obesity is associated with increased risk of hypertension,cardiovascular disease, diabetes, and cancer as well as respiratorycomplications and osteoarthritis [Kopelman (2000) Nature 404:635-643].Even modest weight loss ameliorates these associated conditions.

Maintenance of weight gain or loss is associated with compensatorychanges in energy expenditure that oppose the maintenance of a bodyweight that is different from the usual weight [Leibel et al. (1995) NEngl J Med 332:621-8]. These changes may account, in part, for the poorlong-term efficacy of obesity treatments [Wadden (1993) Ann Intern Med229:688-93]. Further, the decreased insulin sensitivity after weightgain and the beneficial effects of even modest amounts of weightreduction on carbohydrate metabolism and insulin sensitivity in somepatients are well documented [Olefsky et al. (1974) J Clin Invest53:64-76].

While still acknowledging that lifestyle factors including environmentdiet, age and exercise play a role in obesity, twin studies, analyses offamilial aggregation, and adoption studies all indicate that obesity islargely the result of genetic factors [Barsh et al. (2000) Nature404:644-651]. In agreement with these studies, is the fact that anincreasing number of obesity-related genes are being identified. Some ofthe more extensively studied genes include those encoding leptin (ob)and its receptor (db), pro-opiomelanocortin (Pomc),melanocortin-4-receptor (Mc4r), agouti protein (A^(y)), carboxypeptidaseE (fat), 5-hydroxytryptamine receptor 2C (Htr-2c), nescient basichelix-loop-helix 2 (Nhlh2), prohormone convertase 1 (PCSK1), and tubbyprotein (tubby) [rev'd in Barsh et al. (2000) Nature 404:644-651].

SUMMARY OF THE INVENTION

The instant invention is based on the discovery that fragments of thefull-length OBG3 polypeptide comprising the globular domain, termedgOBG3 polypeptide fragments, form homotrimers having unexpected effectsin vitro and in vivo, including utility for weight reduction, preventionof weight gain, and control of blood glucose levels in humans and othermammals. The invention is further based on the discovery that multimersof gOBG3 homotrimer formed through disulfide linkage at the cysteineresidue within the N-terminally disposed unique region have lowerspecific activity for the activities disclosed herein than doesnon-multimeric gOBG3 homotrimer. The instant invention is yet furtherbased on the discovery that gOBG3 polypeptide fragments comprising allor part of the collagen-like region form more stable gOBG3 homotrimershaving the activities disclosed herein.

These unexpected effects of homotrimeric gOBG3 polypeptide fragmentadministration in mammals also include reduction of elevated free fattyacid levels caused by administration of epinephrine, i.v. injection of“intralipid”, or administration of a high fat test meal, as well asincreased fatty acid oxidation in muscle cells, and weight reduction inmammals consuming a high fat/high sucrose diet. These effects areunexpected and surprising given that administration of multimers ofgOBG3 homotrimer typically has no effect or a significantly reducedeffect in vivo or in vitro depending on the specific biological activityand the amount administered. To the extent that any effect is observedfollowing administration of multimers of gOBG3 homotrimer, the levels ofmultimeric gOBG3 homotrimer required for an effect render it unfeasiblein most instances as a potential treatment for humans at this time. Incontrast, non-multimeric gOBG3 homotrimer of the invention is radicallymore effective and thus can be provided at levels that are feasible fortreatments in humans.

Thus, the invention is drawn to gOBG3 polypeptide fragments,polynucleotides encoding said gOBG3 polypeptide fragments, vectorscomprising said gOBG3 polynucleotides, and cells recombinant for saidgOBG3 polynucleotides, as well as to pharmaceutical and physiologicallyacceptable compositions comprising said gOBG3 polypeptide fragments andmethods of administering said gOBG3 pharmaceutical and physiologicallyacceptable compositions in order to reduce body weight or to treatobesity-related diseases and disorders, wherein said gOBG3 polypeptidefragment comprises all or part of the collagen-like region, and furtherwherein said gOBG3 polypeptide fragment does not comprise the cysteineresidue within the N-terminally disposed unique region either becausesaid cysteine residue has been substituted with an amino acid other thancysteine or because said fragment does not span said cysteine residue.Assays for identifying agonists and antagonists of obesity-relatedactivity are also part of the invention.

Antagonists of homotrimeric gOBG3 polypeptide fragment activity shouldbe effective in the treatment of other metabolic-related diseases ordisorders of the invention including cachexia, wasting, AIDS-relatedweight loss, cancer-related weight loss, anorexia, and bulimia. Inpreferred embodiments, said individual is a mammal, preferably a human.

In a first aspect, the invention features a purified, isolated, orrecombinant gOBG3 polypeptide fragment, wherein said gOBG3 polypeptidefragment forms homotrimers having unexpected activity, whereinunexpected said activity is selected from the group consisting of lipidpartitioning, lipid metabolism, and insulin-like activity, wherein saidgOBG3 polypeptide comprises all or part of the collagen-like region, andwherein said gOBG3 polypeptide fragment comprises a substitution of anamino acid other than cysteine for the cysteine within the N-terminallydisposed unique region selected from the group consisting of alanine,aspartic acid, glutamic acid, phenylalanine, glycine, histidine,isoleucine, lysine, leucine, methionine, asparagines, proline,glutamine, arginine, serine, threonine, valine, tryptophan, andtyrosine, preferably wherein said substituted amino acid is serine. Inpreferred embodiments, said polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 and not more than 243consecutive amino acids of SEQ ID NO:2 wherein said polypeptide fragmentcomprises all or part of the collagen-like region, and wherein thecysteine at position 36 is replaced by said substitute amino acid; or atleast 6 and not more than 246 consecutive amino acids of SEQ ID NO:4wherein said polypeptide comprises all or part of the collagen-likeregion and wherein the cysteine at position 39 is replaced by saidsubstitute amino acid. In other preferred embodiments, gOBG3 polypeptidefragments having unexpected activity are selected from amino acids18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244,27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244,36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2wherein the cysteine at position 36 is replaced by said substitute aminoacid. In other preferred embodiments, gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 18-247, 19-247,20-247, 21-247, 22-247,23-247, 24-247, 25-247, 26-247, 27-247, 28-247,29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-246, 36-247, 37-247,38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQID NO:4, wherein the cysteine at position 39 is replaced by saidsubstitute amino acid. In most preferred embodiments, gOBG3 polypeptidefragments having unexpected activity are human. In other furtherpreferred embodiments, said polypeptide fragment comprises an amino acidsequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identical to the corresponding consecutive amino acids of SEQ IDNO:2 or SEQ ID NO:4.

In other highly preferred embodiments, the invention features an OBG3polypeptide fragment wherein said OBG3 polypeptide fragment formshomotrimers having unexpected activity selected from the groupconsisting of lipid partitioning, lipid metabolism, and insulin-likeactivity, wherein said OBG3 polypeptide fragment comprises, consistsessentially of, or consists of a purified, isolated, or recombinantgOBG3 polypeptide fragment, wherein said gOBG3 polypeptide fragmentcomprises all or part of the collagen-like region, and wherein saidgOBG3 polypeptide fragment comprises a substitution of an amino acidother than cysteine for the cysteine within the N-terminally disposedunique region selected from the group consisting of alanine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, leucine, methionine, asparagines, proline, glutamine, arginine,serine, threonine, valine, tryptophan, and tyrosine, preferably whereinsaid substituted amino acid is serine. In preferred embodiments, gOBG3polypeptide fragments having unexpected activity are selected from aminoacids 18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244,26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244,35-244, 36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQID NO:2, wherein the cysteine at position 36 is replaced by saidsubstitute amino acid. In other preferred embodiments, gOBG3 polypeptidefragments having unexpected activity are selected from amino acids18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247, 26-247,27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247,36-247, 37-247-38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247,or 45-247 of SEQ ID NO:4, wherein the cysteine at position 39 isreplaced by said substitute amino acid. In most preferred embodiments,gOBG3 polypeptide fragments having unexpected activity are human.Alternatively, said gOBG3 polypeptide fragment comprises, consistsessentially of, or consists of, an amino acid sequence at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe corresponding amino acids 18-244 of SEQ ID NO:2 or at least 75%identical to amino acids 18-247 of SEQ ID NO:4.

In other highly preferred embodiments, the invention features apurified, isolated, or recombinant gOBG3 polypeptide fragment, whereinsaid gOBG3 polypeptide fragment forms homotrimers having unexpectedactivity, wherein unexpected said activity is selected from the groupconsisting of prevention of weight gain, weight reduction, andmaintenance of weight loss, wherein said gOBG3 polypeptide fragmentcomprises all or part of the collagen-like region, and wherein saidgOBG3 polypeptide fragment comprises a substitution of an amino acidother than cysteine for the cysteine within the N-terminally disposedunique region selected from the group consisting of alanine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, leucine, methionine, asparagines, proline, glutamine, arginine,serine, threonine, valine, tryptophan, and tyrosine, preferably whereinsaid substituted amino acid is serine. In preferred embodiments, saidpolypeptide fragment comprises, consists essentially of, or consists of,at least 6 and not more than 243 consecutive amino acids of SEQ ID NO:2wherein said gOBG3 polypeptide fragment comprises all or part of thecollagen-like region and wherein the cysteine at position 36 is replacedby said substitute amino acid or at least 6 and not more than 246consecutive amino acids of SEQ ID NO:4 wherein said gOBG3 polypeptidefragment comprises all or part of the collagen-like region and whereinthe cysteine at position 39 is replaced by said substitute amino acid.In other preferred embodiments, gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 18-244, 19-244,20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244, 27-244, 28-244,29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244, 36-244, 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2 wherein thecysteine at position 36 is replaced by said substitute amino acid. Inother preferred embodiments, gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 18-247, 19-247,20-247, 21-247, 22-247, 23-247, 24-247, 25-247, 26-247, 27-247, 28-247,29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247, 36-247, 37-247,38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQID NO:4, wherein the cysteine at position 39 is replaced by saidsubstitute amino acid. In most preferred embodiments, gOBG3 polypeptidefragment having unexpected activity is human. In other further preferredembodiments, said polypeptide fragment comprises an amino acid sequenceat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%identical to the corresponding consecutive amino acids of SEQ ID NO:2 orSEQ ID NO:4.

In other highly preferred embodiments, the invention features an OBG3polypeptide fragment wherein said OBG3 polypeptide fragment formshomotrimers having unexpected activity selected from the groupconsisting of prevention of weight gain, weight reduction, andmaintenance of weight loss and wherein said polypeptide fragmentcomprises, consists essentially of, or consists of a purified, isolated,or recombinant gOBG3 polypeptide fragment, wherein said gOBG3polypeptide fragment comprises all or part of the collagen-like region,and wherein said gOBG3 polypeptide fragment comprises a substitution ofan amino acid other than cysteine for the cysteine within theN-termially disposed unique region selected from the group consisting ofalanine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagines,proline, glutamine, arginine, serine, threonine, valine, tryptophan, andtyrosine, preferably wherein said substituted amino acid is serine.Preferably, said gOBG3 polypeptide fragment comprises, consistsessentially of, or consists of, at least 6 consecutive amino acids ofamino acids 18 to 244 of SEQ ID NO:2 wherein said gOBG3 polypeptidefragment comprises all or part of the collagen-like region and whereinthe cysteine at position 36 is replaced by said substitute amino acid orat least 6 consecutive amino acids of amino acids 18 to 247 of SEQ IDNO:4 wherein said gOBG3 polypeptide fragment comprises all or part ofthe collagen-like region wherein the cysteine at position 39 is replacedby said substitute amino acid. In other preferred embodiments, gOBG3polypeptide fragments having unexpected activity are selected from aminoacids 18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244,26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244,35-244, 36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQID NO:2 wherein the cysteine at position 36 is replaced by saidsubstitute amino acid. In other preferred embodiments, gOBG3 polypeptidefragments having unexpected activity are selected from amino acids18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247, 26-247,27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247,36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247,or 45-247 of SEQ ID NO:4 wherein the cysteine at position 39 is replacedby said substitute amino acid. In most preferred embodiments, gOBG3polypeptide fragment having unexpected activity is human. Alternatively,said gOBG3 fragment comprises, consists essentially of, or consists ofan amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the corresponding amino acids 18-244of SEQ ID NO:2 or at least 75% identical to amino acids 18-247 of SEQ DNO:4.

In yet other highly preferred embodiments, the invention features apurified, isolated, or recombinant gOBG3 polypeptide fragment, whereinsaid gOBG3 polypeptide fragment forms homotrimers having unexpectedactivity, wherein unexpected said activity is selected from the groupconsisting of lipid partitioning, lipid metabolism, and insulin-likeactivity, wherein said gOBG3 polypeptide fragment comprises all or partof the collagen-like region, and wherein said gOBG3 polypeptide fragmentdoes not span the cysteine residue within the N-terminally disposedunique region. In preferred embodiments, said polypeptide fragmentcomprises, consists essentially of, or consists of, at least 6 and notmore than 243 consecutive amino acids of SEQ ID NO:2 or at least 6 andnot more than 246 consecutive amino acids of SEQ ID NO:4. In otherpreferred embodiments, gOBG3 polypeptide fragments having unexpectedactivity are selected from amino acids 37-244, 38-244, 39-244, 40-244,41-244, or 42-244 of SEQ ID NO:2. In other preferred embodiments, gOBG3polypeptide fragments having unexpected activity are selected from aminoacids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4.In most preferred embodiments, gOBG3 polypeptide fragment havingunexpected activity is human. In other further preferred embodiments,said polypeptide fragment comprises an amino acid sequence at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe corresponding consecutive amino acids of SEQ ID NO:2 or SEQ ID NO:4.

In other highly preferred embodiments, the invention features an OBG3polypeptide fragment wherein said OBG3 polypeptide fragment formshomotrimers having unexpected activity selected from the groupconsisting of lipid partitioning, lipid metabolism, and insulin-likeactivity, wherein said OBG3 polypeptide fragment comprises, consistsessentially of, or consists of a purified, isolated, or recombinantgOBG3 polypeptide fragment, wherein said gOBG3 polypeptide fragmentcomprises all or part of the collagen-like region and wherein said gOBG3polypeptide fragment does not span the cysteine residue within theN-terminally disposed unique region. In preferred embodiments, gOBG3polypeptide fragments having unexpected activity are selected from aminoacids 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2.In other preferred embodiments, gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 40-247, 41-247,42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4. In most preferredembodiments, gOBG3 polypeptide fragment having unexpected activity ishuman. Alternatively, said gOBG3 polypeptide fragment comprises,consists essentially of, or consists of, an amino acid sequence at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the corresponding amino acids 37-244 of SEQ ID NO:2 or at least 75%identical to amino acids 40-247 of SEQ ID NO:4.

In other highly preferred embodiments, the invention features apurified, isolated, or recombinant gOBG3 polypeptide fragment, whereinsaid gOBG3 polypeptide fragment forms homotrimers having unexpectedactivity, wherein unexpected said activity is selected from the groupconsisting of prevention of weight gain, weight reduction, andmaintenance of weight loss, wherein said gOBG3 polypeptide fragmentcomprises all or part of the collagen-like region and wherein said gOBG3polypeptide fragment does not span the cysteine residue within theN-terminally disposed collagen region. In preferred embodiments, saidpolypeptide fragment comprises, consists essentially of, or consists of,at least 6 and not more than 243 consecutive amino acids of SEQ ID NO:2or at least 6 and not more than 246 consecutive amino acids of SEQ IDNO:4. In other preferred embodiments, gOBG3 polypeptide fragments havingunexpected activity are selected from amino acids 37-244, 38-244,39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2. In other preferredembodiments, gOBG3 polypeptide fragments having unexpected activity areselected from amino acids 40-247, 41-247, 42-247, 43-247, 44-247, or45-247 of SEQ ID NO:4. In most preferred embodiments, gOBG3 polypeptidefragment having unexpected activity is human. In other further preferredembodiments, said polypeptide fragment comprises an amino acid sequenceat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding consecutive amino acids of SEQ ID NO:2 orSEQ ID NO:4.

In other highly preferred embodiments, the invention features an OBG3polypeptide fragment wherein said OBG3 polypeptide fragment formshomotrimers having unexpected activity selected from the groupconsisting of prevention of weight gain, weight reduction, andmaintenance of weight loss and wherein said polypeptide fragmentcomprises, consists essentially of, or consists of a purified, isolated,or recombinant gOBG3 polypeptide fragment, wherein said gOBG3polypeptide fragment comprises all or part of the collagen-like regionand wherein said gOBG3 polypeptide fragment does not span the cysteineresidue within the N-terminally disposed unique region. Preferably, saidgOBG3 polypeptide fragment comprises, consists essentially of, orconsists of, at least 6 consecutive amino acids of amino acids 37 to 244of SEQ ID NO:2 comprising all or part of the collagen-like region or atleast 6 consecutive amino acids of amino acids 40 to 247 of SEQ ID NO:4comprising all or part of the collagen-like region. In other preferredembodiments, gOBG3 polypeptide fragments having unexpected activity areselected from amino acids 37-244, 38-244, 39-244, 40-244, 41-244, or42-244 of SEQ ID NO:2. In other preferred embodiments, gOBG3 polypeptidefragments having unexpected activity are selected from amino acids40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4. Inmost preferred embodiments, gOBG3 polypeptide fragment having unexpectedactivity is human. Alternatively, said gOBG3 fragment comprises,consists essentially of, or consists of, an amino acid sequence at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the corresponding amino acids 37-244 of SEQ ID NO:2 or at least 75%identical to amino acids 40-247 of SEQ ID NO:4.

In a further preferred embodiment, the gOBG3 polypeptide fragment formshomotrimers able to lower circulating (either blood, serum or plasma)levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or(iii) triglycerides. Further preferred gOBG3 polypeptide fragments formhomotrimers that demonstrate free fatty acid level lowering activity,glucose level lowering activity, and/or triglyceride level loweringactivity, have an activity that is significantly greater thanfull-length OBG3 at the same molar concentration, have a greater thantransient activity and/or have a sustained activity.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that maintain weight loss, preferably in individuals whowere previously “obese” and are now “healthy” (as defined herein).

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that significantly stimulate muscle lipid or free fatty acidoxidation as compared to full-length OBG3 polypeptides at the same molarconcentration. Further preferred gOBG3 polypeptide fragments are thosethat form homotrimers that cause C2C12 cells differentiated in thepresence of said fragments to undergo at least 10%, 20%, 30%, 35%, or40% more oleate oxidation as compared to untreated cells or cellstreated with full-length OBG3.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that are at least 30% more efficient than full-length OBG3at increasing leptin uptake in a liver cell line (preferably BPRCL mouseliver cells (ATCC CRL-2217)).

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that significantly reduce the postprandial increase inplasma free fatty acids, particularly following a high fat meal.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that significantly reduce or eliminate ketone bodyproduction, particularly following a high fat meal.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase glucose uptake in skeletal muscle cells.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase glucose uptake in adipose cells.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase glucose uptake in neuronal cells.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase glucose uptake in red blood cells.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase glucose uptake in the brain.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that significantly reduce the postprandial increase inplasma glucose following a meal, particularly a high carbohydrate meal.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that significantly prevent the postprandial increase inplasma glucose following a meal, particularly a high fat or a highcarbohydrate meal.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that improve insulin sensitivity.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that inhibit the progression from impaired glucose toleranceto insulin resistance.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that increase muscle mass, preferably those that increasemuscle cell number, more preferably those that increase muscle fibernumber.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that promote an increase in body girth, preferably fragmentsthat promote an increase in muscle mass. Further preferred gOBG3polypeptide fragments promote growth rate, preferably promoting anincrease in growth rate greater than an average growth rate in theabsence of gOBG3 polypeptide fragments.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers that promote growth rate in newborn mammals, preferably cow,goat, sheep, rabbit, mouse, rat, pig, dog, or human newborns, morepreferably human newborns between the ages of 0-6 months of age, mostpreferably human newborn between the ages of 0-3 months. Furtherpreferred gOBG3 polypeptide fragments are those that form homotrimersthat promote growth rate in newborn underweight or premature mammals,preferably cow, goat, sheep, rabbit, mouse, rat, pig, dog, or humanunderweight or premature newborns, more preferably human underweight orpremature newborns between the ages of 0-6 months of age, mostpreferably human underweight or premature newborns between the ages of0-3 months of age.

Further preferred gOBG3 polypeptide fragments are those that formhomotrimers in vitro and/or in vivo. More preferred gOBG3 polypeptidefragments are those that form homotrimers in vitro and/or in vivo,wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of said gOBG3 polypeptide fragment comprises saidhomotrimer. Most particularly preferred gOBG3 polypeptide fragments arethose that form homotrimers in vitro and/or in vivo having activityselected from the group consisting of lipid partitioning, lipidmetabolism, and insulin-like activity. Also most particularly preferredgOBG3 polypeptide fragments are those that form homotrimers in vitroand/or in vivo having activity selected from the group consisting ofprevention of weight gain, weight reduction, and maintenance of weightloss.

Further preferred embodiments include heterologous polypeptidescomprising a gOBG3 polypeptide fragment of the invention. More preferredis said heterologous polypeptide comprised of a signal peptideN-terminally fused to said gOBG3 polypeptide of the invention. In yetmore preferred embodiment, said signal peptide is human zinc-alpha2-glycoprotein signal peptide of amino acid sequenceMVRMVPVLLSLLLLLGPAVP, preferably encoded by the polynucleotide ofsequence atggtaagaatggtgcctgtcctgctgtctctgctgctgcttctgggtcctgctgtcccc.

In a second aspect, the invention features a purified, isolated, orrecombinant polynucleotide encoding said gOBG3 polypeptide fragment orfull-length OBG3 polypeptide described in the first aspect, or thecomplement thereof In further embodiments the polynucleotides are DNA,RNA, DNA/RNA hybrids, single-stranded, and double-stranded.

In a third aspect the invention features a recombinant vectorcomprising, consisting essentially of, or consisting of, saidpolynucleotide described in the second aspect.

In a fourth aspect, the invention features a recombinant cellcomprising, consisting essentially of, or consisting of, saidrecombinant vector descnbed in the third aspect. Preferred saidrecombinant cell is prokaryotic or eukaryotic recombinant cell.Preferred said prokaryotic recombinant cell is E. coli recombinant cell.Preferred said eukaryotic recombinant cell is mammalian recombinantcell. Particularly preferred mammalian recombinant cell is selected fromthe group consisting of COS recombinant cell, Chinese hamster ovary(CHO) recombinant cell, human embryonic kidney (HEK) recombinant cell,and 3T3-L1 adipocyte recombinant cell. A further embodiment includes ahost cell recombinant for a polynucleotide of the invention. Preferredsaid host cell is prokaryotic or eukaryotic host cell. Preferred saidprokaryotic host cell is E. coli host cell. Preferred said eukaryotichost cell is mammalian host cell. Particularly preferred mammalian hostcell is selected from the group consisting of COS host cell, Chinesehamster ovary (CHO) host cell, human embryonic kidney (HEK) host cell,and 3T3-L1 adipocyte host cell.

In a fifth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of, said gOBG3 polypeptide fragmentdescribed in the first aspect and, alternatively, a pharmaceutical orphysiologically acceptable diluent. More preferred said pharmaceuticalor physiologically acceptable composition comprises, consistsessentially of, or consists of homotrimer of said gOBG3 polypeptidefragment described in the first aspect and, alternatively, apharmaceutical or physiologically acceptable diluent. In saidpharmaceutical or physiologically acceptable composition, preferably atleast 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of said gOBG3 polypeptide fragment of the first aspect compriseshomotrimer.

In a sixth aspect, the invention features a method of reducing body masscomprising providing or administering to individuals in need of reducingbody mass said pharmaceutical or physiologically acceptable compositiondescribed in the fifth aspect. Further preferred is a method of reducingbody fat mass comprising providing or administering to individuals inneed thereof said pharmaceutical or physiologically acceptablecomposition described in the fifth aspect. Further preferred is a methodof increasing lean body mass comprising providing or administering toindividuals in need thereof said pharmaceutical or physiologicallyacceptable composition described in the fifth aspect. Further preferredis a method of increasing the growth rate of body girth or lengthcomprising providing or administering to individuals in need thereofsaid pharmaceutical or physiologically acceptable composition describedin the fifth aspect.

In a further preferred embodiment, the present invention may be used incomplementary therapy of obese patients to improve their weight incombination with a weight reducing agent. Examples of the weightreducing agent include lipase inhibitors, such as orlistat, andserotonin reuptake inhibitors (SSRI) and noradrenaline reuptakeinhibitor, such as sibutramine.

In further preferred embodiments, the invention features a method ofmaintaining a reduced body mass comprising providing or administering toindividuals in need of maintaining a reduced body mass saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect. Further preferred is a method of maintaining a reducedbody fat mass that comprises, providing or administering to individualsin need thereof said pharmaceutical or physiologically acceptablecomposition described in the fifth aspect, returning energy intake to anormal level in said individual, and maintaining increased energyexpenditure in said individual. Preferrably, said individual is able tomaintain a stable weight that is 10-20% below their obese weight (asdescribed herein).

In other preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used incombination with reduced energy intake and/or increased energyexpenditure as a method of maintaining weight loss.

In yet further preferred embodiments, the identification of saidindividuals in need of reducing body mass to be treated with saidpharmaceutical or physiologically acceptable composition comprisesgenotyping OBG3 single nucleotide polymorphisms (SNPs) or measuring OBG3polypeptide or mRNA levels in clinical samples from said individuals.Preferably, said clinical samples are selected from the group consistingof plasma, urine, and saliva. Preferably, a gOBG3 polypeptide fragmentof the present invention is administered to an individual with at leasta 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction inblood, serum or plasma levels of full-length OBG3 or the naturallyproteolytically cleaved OBG3 fragment as compared to healthy, non-obesepatients.

In a seventh aspect, the invention features a method of preventing ortreating an obesity-related disease or disorder comprising providing oradministering to an individual in need of such treatment saidpharmaceutical or physiologically acceptable composition descnbed in thefifth aspect. In preferred embodiments, the identification of saidindividuals in need of such treatment to be treated with saidpharmaceutical or physiologically acceptable composition comprisesgenotyping OBG3 single nucleotide polymorphisms (SNPs) or measuring OBG3polypeptide or mRNA levels in clinical samples from said individuals.Preferably, said clinical samples are selected from the group consistingof blood, serum, plasma, urine, and saliva. Preferably, saidobesity-related disease or disorder is selected from the groupconsisting of obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother metabolic-related diseases or disorders of the invention includingcachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia. In preferred embodiments, said individual is amammal, preferably a human.

In related aspects, embodiments of the present invention includesmethods of causing or inducing a desired biological response in anindividual comprising the steps of: providing or administering to anindividual a composition comprising a polypeptide of the first aspect,wherein said biological response is selected from the group consistingof:

(a) lowering circulating (either blood, serum, or plasma) levels(concentration) of free fatty acids;

(b) lowering circulating (either blood, serum or plasma) levels(concentration) of glucose;

(c) lowering circulating (either blood, serum or plasma) levels(concentration) of triglycerides;

(d) stimulating muscle lipid or free fatty acid oxidation;

(c) increasing leptin uptake in the liver or liver cells;

(e) reducing the postprandial increase in plasma free fatty acids,particularly following a high fat meal; and,

(f) reducing or eliminating ketone body production, particularlyfollowing a high fat meal;

(g) increasing tissue sensitivity to insulin, particularly muscle,adipose, liver or brain;

(h) inhibiting the progression from impaired glucose tolerance toinsulin resistance;

(i) increasing muscle cell protein synthesis;

(j) reducing adipocyte triglyceride content;

(k) increasing utilization of energy from foodstuffs or metabolicstores;

(l) increasing growth rate, preferably growth in girth or length;

(m) increasing muscle growth; and

(n) increasing skeletal growth;

and further wherein said biological response is significantly greaterthan, or at least 10%, 20%, 30%, 35%, or 40% greater than, thebiological response caused or induced by a full-length OBG3 polypeptideat the same molar concentration; or alternatively wherein saidbiological response is greater than a transient response; oralternatively wherein said biological response is sustained. In furtherpreferred embodiments, the present invention of said pharmaceutical orphysiologically acceptable composition can be used as a method tocontrol blood glucose in some persons with Noninsulin Dependent DiabetesMellitus (NIDDM, Type II diabetes) in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) in combination with insulintherapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, withoutcombination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some persons with Insulin DependentDiabetes Mellitus (IDDM, Type I diabetes) alone, without combination ofinsulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy of NIDDM patients to improve their weight orglucose control in combination with an oral insulin secretagogue or aninsulin sensitising agent. Preferably, the oral insulin secretagogue is1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of NIDDM patients alone, without an oralinsulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may be used incomplementary therapy of IDDM patients to improve their weight orglucose control in combination with an oral insulin secretagogue or aninsulin sensitising agent. Preferably, the oral insulin secretagogue is1,1-dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or asulphonylurea selected from tolbutamide, tolazamide, chlorpropamide,glibenclamide, glimepiride, glipizide and glidazide. Preferably, theinsulin sensitising agent is selected from metformin, ciglitazone,troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of IDDM patients alone, without an oralinsulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the oral insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an oral insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin NIDDM or IDDM patients.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) in combinationwith insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with InsulinDependent Diabetes Mellitus (IDDM, Type I diabetes) in combination withinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some persons with NoninsulinDependent Diabetes Mellitus (NIDDM, Type II diabetes) without insulintherapy.

In an eighth aspect, the invention features a method of making the gOBG3polypeptide fragment described in the first aspect, wherein said methodis selected from the group consisting of: proteolytic cleavage,recombinant methodology and artificial synthesis.

In a ninth aspect, the present invention provides a method of making arecombinant gOBG3 polypeptide fragment or a full-length OBG3polypeptide, the method comprising providing a transgenic, non-humanmammal whose milk contains said recombinant gOBG3 polypeptide fragmentor full-length protein, and purifying said recombinant gOBG3 polypeptidefragment or said full-length OBG3 polypeptide from the milk of saidnon-human mammal. In one embodiment, said non-human mammal is a cow,goat, sheep, rabbit, or mouse. In another embodiment, the methodcomprises purifying a recombinant mature OBG3 polypeptide absent thesignal peptide from said milk, and further comprises cleaving saidprotein in vitro to obtain a desired gOBG3 polypeptide fragment In atenth aspect, the invention features a use of the polypeptide describedin the first aspect for treatment of obesity-related diseases anddisorders and/or reducing body mass. Preferably, said obesity-relateddiseases and disorders are selected from the group consisting ofobesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Inpreferred embodiments, said individual is a mammal, preferably a human.

The invention further features a use of the polypeptide of the firstaspect for prevention of weight gain, for weight reduction, and/or formaintenance of weight loss. In preferred embodiments, said individual isa mammal, preferably a human.

In an eleventh aspect, the invention features a use of the polypeptidedescribed in the first aspect for the preparation of a medicament forthe treatment of obesity-related diseases and disorders and/or forreducing body mass. Preferably, said obesity-related disease or disorderis selected from the group consisting of obesity, impaired glucosetolerance, insulin resistance, atherosclerosis, atheromatous disease,heart disease, hypertension, stroke, Syndrome X, Noninsulin DependentDiabetes Mellitus (NIDDM, or Type II diabetes) and Insulin DependentDiabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other metabolic-relateddiseases or disorders of the invention including cachexia, wasting,AIDS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human.

The invention further features a use of the polypeptide of the firstaspect for the preparation of a medicament for prevention of weightgain, for weight reduction, and/or for maintenance of weight loss. Inpreferred embodiments, said individual is a mammal, preferably a human.

In a twelfth aspect, the invention provides a polypeptide of the firstaspect of the invention, or a composition of the fifth aspect of theinvention, for use in a method of treatment of the human or animal body.

In a thirteenth aspect, the invention features methods of reducing bodyweight comprising providing to an individual said pharmaceutical orphysiologically acceptable composition described in the fifth aspect, orthe polypeptide described in the first aspect. Where the reduction ofbody weight is practiced for cosmetic purposes, the individual has a BMIof at least 20 and no more than 25. In embodiments for the treatment ofobesity, the individual may have a BMI of at least 20. One embodimentfor the treatment of obesity provides for the treatment of individualswith BMI values of at least 25. Another embodiment for the treatment ofobesity provides for the treatment of individuals with BMI values of atleast 30. Yet another embodiment provides for the treatment ofindividuals with BMI values of at least 40. Alternatively, forincreasing the body weight of an individual, the BMI value should be atleast 15 and no more than 20.

In a related aspect, the invention features methods of maintainingweight loss comprising providing to an individual said pharmaceutical orphysiologically acceptable composition described in the fifth aspect, orthe polypeptide described in the first aspect. Where the maintenance ofweight loss is practiced for cosmetic purposes, the individual has a BMIof at least 20 and no more than 25. In embodiments for the treatment ofobesity by means of maintaining weight loss, the individual may have aBMI of at least 20. One embodiment for the treatment of obesity by meansof maintaining weight loss provides for the treatment of individualswith BMI values of at least 25. Another embodiment for the treatment ofobesity by means of maintaining weight loss provides for the treatmentof individuals with BMI values of at least 30.

In a fourteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body mass and/or for treatment or prevention of obesity-relateddiseases or disorders. Preferably, said obesity-related disease ordisorder is selected from the group consisting of obesity, impairedglucose tolerance, insulin resistance, atherosclerosis, atheromatousdisease, heart disease, hypertension, stroke, Syndrome X, NoninsulinDependent Diabetes Mellitus (NIDDM, or Type II diabetes) and InsulinDependent Diabetes Mellitus (IDDM or Type I diabetes). Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy, andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia and hyperuricemia. Yet other obesity-relateddiseases or disorders of the invention include cachexia, wasting,AIDS-related weight loss, cancer-related weight loss, anorexia, andbulimia. In preferred embodiments, said individual is a mammal,preferably a human. In preferred embodiments, the identification of saidindividuals to be treated with said pharmaceutical or physiologicallyacceptable composition comprises genotyping OBG3 single nucleotidepolymorphisms (SNPs) or measuring OBG3 polypeptide or mRNA levels inclinical samples from said individuals. Preferably, said clinicalsamples are selected from the group consisting of blood, serum, plasma,urine, and saliva.

In a fifteenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect forreducing body weight for cosmetic reasons.

In a related aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect formaintaining weight loss for cosmetic reasons.

In a sixteenth aspect, the invention features methods of treatinginsulin resistance comprising providing to an individual saidpharmaceutical or physiologically acceptable composition described inthe fifth aspect, or the polypeptide described in the first aspect.

In a seventeenth aspect, the invention features the pharmaceutical orphysiologically acceptable composition described in the fifth aspect ina method of treating individuals with normal glucose tolerance (NGT) whoare obese or who have fasting hyperinsulinemia, or who have both.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with gestationaldiabetes. Gestational diabetes refers to the development of diabetes inan individual during pregnancy, usually during the second or thirdtrimester of pregnancy.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating individuals with impairedfasting glucose (IFG). Impaired fasting glucose (IFG) is that conditionin which fasting plasma glucose levels in an individual are elevated butnot diagnostic of overt diabetes, i.e. plasma glucose levels of lessthan 126 mg/dl and greater than or equal to 110 mg/dl.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating impaired glucose tolerance(IGT) in an individual. In other further preferred embodiments, theinvention features the pharmaceutical or physiologically acceptablecomposition described in the fifth aspect in a method of preventing IGTin an individual. By providing therapeutics and methods for reducing orpreventing IGT, i.e., for normalizing insulin resistance, theprogression to NIDDM can be delayed or prevented. Furthermore, byproviding therapeutics and methods for reducing or preventing insulinresistance, the invention provides methods for reducing and/orpreventing the appearance of Insulin-Resistance Syndrome.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having polycysticovary syndrome (PCOS). PCOS is among the most common disorders ofpremenopausal women. Insulin-sensitizing agents have been shown to beeffective in PCOS. Accordingly, the invention provides methods forreducing insulin resistance, normalizing blood glucose thus treatingand/or preventing PCOS.

In further preferred embodiments, the invention features thepharmaceutical or physiologically acceptable composition described inthe fifth aspect in a method of treating a subject having insulinresistance. In still further preferred embodiments, a subject havinginsulin resistance is treated according to the methods of the inventionto reduce or cure the insulin-resistance. As insulin resistance is alsooften associated with infections and cancer, prevention or reducinginsulin resistance according to the methods of the invention may preventor reduce infections and cancer.

In further preferred embodiment, the methods of the invention are usedto prevent the development of insulin resistance in a subject, e.g.,those known to have an increased risk of developing insulin-resistance.

In an eighteenth aspect, the invention features a method of usinghomotrimeric gOBG3 polypeptide fragment in a method of screeningcompounds for one or more antagonists of homotrimeric gOBG3 polypeptidefragment activity, wherein said activity is selected from but notrestricted to weight reduction, maintenance of weight loss, lipidpartitioning, lipid metabolism, and insulin-like activity.

In preferred embodiment, said compound is selected from but is notrestricted to small molecular weight organic or inorganic compound,protein, peptide, carbohydrate, or lipid.

In a nineteenth aspect, the present invention provides a mammal,preferably a newborn human, with a supplement to promote, improve,enhance or increase the assimilation, utilization or storage of energyand other nutrients present in foodstuffs consumed by newborn mammals,particularly newborn humans, and particularly energy and other nutrientsin infant formula or breast milk. A further preferred embodiment of thepresent invention is to provide a mammal, preferably a newborn human,with a supplement to promote, improve, enhance, or increase growth rate.Preferred methods of supplementation with a polypeptide of the firstaspect include but are not limited to:

(a) direct addition of a polypeptide of the first aspect to syntheticinfant formula or to breast milk;

(b) administration of the pharmaceutical or physiologically acceptablecomposition described in the fifth aspect prior to feeding, preferably1-15 minutes prior to feeding, more preferably 1-5 minutes prior tofeeding; and

(c) administration of the pharmaceutical or physiologically acceptablecomposition described in the fifth aspect following feeding, preferably1-15 minutes following feeding, more preferably 1-5 minutes followingfeeding;

wherein routes of administration of a polypeptide of the first aspect orthe pharmaceutical or physiologically acceptable composition describedin the fifth aspect are selected from oral, buccal, nasal andintramuscular routes, preferably oral routes.

A further preferred embodiment is directed to using a polypeptide of thefirst aspect in methods to promote, improve, enhance or increase theassimilation, utilization or storage of energy and other nutrientspresent in foodstuffs consumed by newborn mammals, particularly newbornhumans, and particularly energy and other nutrients in infant formula orbreast milk. A further preferred embodiment is directed to using apolypeptide of the first aspect in methods to promote, improve, enhanceor increase the growth rate of a newborn mammal, preferably a humannewborn. Further preferred are compositions comprising a polypeptide ofthe first aspect which can be used in methods to promote, improve,enhance or increase the assimilation, utilization or storage of energyand other nutrients present in foodstuffs consumed by newborn mammals,particularly newborn humans, and particularly energy and other nutrientsin infant formula or breast milk. Further preferred are compositionscomprising a polypeptide of the first aspect which can be used inmethods to promote, improve, enhance or increase the growth rate of anewborn mammal, preferably a human newborn. A still further preferredembodiment of the present invention is directed to compositionscomprising synthetic infant milk formula and a polypeptide of the firstaspect.

Another embodiment of the invention is to provide compositionscomprising a polypeptide of the first aspect useful for enhancing orimproving the nutritional value of synthetic infant milk formulas orbreast milk. Further preferred are compositions useful for incorporationinto the diet of a newborn mammal so as to enhance and improve thenutritional value of the diet. Still another embodiment of the inventionis to provide techniques and routines for improving the diet and feedingof newborn mammals, particularly premature, underweight orvery-low-birth-weight newborns, preferably human newborns.

In preferred aspects of the methods of the invention disclosed herein,the amount of gOBG3 polypeptide fragment or polynucleotide administeredto an individual is sufficient to bring circulating (blood, serum, orplasma) levels (concentration) of OBG3 polypeptides to their normallevels (levels in non-obese individuals). “Normal levels” may bespecified as the total concentration of all circulating OBG3polypeptides (full-length OBG3 and fragments thereof) or theconcentration of all circulating proteolytically cleaved OBG3polypeptides only.

In preferred embodiments of the compositions of the invention disclosedherein, compositions of the invention may further comprise anycombination of gOBG3 polypeptide fragment of the first aspect, insulin,insulin secretagogues or insulin sensitising agents such that thecomposition produces a biological effect greater than the expectedeffect for said gOBG3 polypeptide fragment administered alone ratherthan in combination with insulin, insulin secretagogues or insulinsensitising agents.

In a further embodiment, said biological function includes, but is notlimited to, free fatty acid level lowering activity, glucose levellowering activity, triglyceride level lowering activity, stimulatingadipose lipolysis, stimulating muscle lipid or free fatty acidoxidation, increasing leptin uptake in a liver cell line, significantlyreducing the postprandial increase in plasma free fatty acids or glucosedue to a high fat meal, significantly reducing or eliminate ketone bodyproduction as the result of a high fat meal, increasing glucose uptakein skeletal muscle cells, adipose cells, red blood cells or the brain,increasing insulin sensitivity, inhibiting the progression from impairedglucose tolerance to insulin resistance, reducing body mass, decreasingfat mass, increasing lean muscle mass, preventing or treating anmetabolic-related disease or disorder, controlling blood glucose in somepersons with Noninsulin Dependent Diabetes Mellitus or NoninsulinDependent Diabetes Mellitus, treating insulin resistance or preventingthe development of insulin resistance.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the sequences of the human (APM1), andmouse (AdipoQ and ACRP30) OBG3 polypeptides.

FIG. 2 shows the nucleic acid sequence of AdipoQ cloned into the BamHIand XhoI sites of pTrcHisB. AdipoQ begins at 510 and ends at 1214(insert in bold). This construct does not contain the N-term signalpeptide-derived sequence (MLLLQALLFLLILP).

FIG. 3 shows a schematic drawing of the protein structure of APM1. Theputative signal sequence at the N-terminus (AA 1-17), the unique region(AA 1841), the collagen-like region (AA 42-107), and the globular domain(AA 108-244) at the carboxy terminus are shown. Two protease cleavagesites after AA 100 and AA 131 are also shown.

FIG. 4 shows the nucleic acid sequence of the globular domain of AdipoQcloned into pTrcHisB. AdipoQ globular domain begins at 510 and ends at927 bp. The insert is in bold.

FIG. 5 is a graph showing a comparison of the effect of AdipoQ (AQ) andAdipoQ globular head (AQ-GH) on cell-associated ¹²⁵I-leptin in the mouseliver cell line BPRCL. Results are shown as percent of control values inthe presence of increasing amounts of compound (AQ or AQ-GH), and arethe mean of triplicate determinations.

FIGS. 6A, 6B, and 6C show graphs of ¹²⁵I-LDL binding, uptake, anddegradation, respectively, in the mouse liver cell line BPRCL in thepresence of increasing amounts of gOBG3.

FIG. 7 shows a protein sequence alignment of the obg3 clone (obg3 clone;the insert in FIG. 2) with the published sequences of human (APM1) andmouse (AdipoQ and ACRP30) obg3. In the alignment, amino acids (AAs) 45to 110 contain the collagen-like region; AAs 111-247 contain theglobular domain. The cut sites from lysine-blocked trypsin fall afterAAs 58, 61, 95, 103, 115, 125, and 134. As determined by amino-terminalsequencing of the gOBG3 product, the gOBG3 start site is at AA 104 (101for human gOBG3 or APM1).

FIG. 8 shows a graphical representation of the effect of gOBG3 (3×25 μgip) on plasma free fatty acids (FFA) in C57BL6/J mice following a highfat meal (*p<0.02).

FIGS. 9A and 9B show graphical representations of the effect of gOBG3(3×25 μg ip) on plasma triglycerides (TG) in C57BL6/J mice following ahigh fat meal (p<0.05 at 2, 3 and 4 hours). FIG. 9A shows TG in mg/dl;FIG. 9B shows TG as a percent of the starting value.

FIG. 10 shows a graphical representation of the effect of gOBG3 (3×25 μgip) on plasma glucose in CS7BL6/J mice following a high fat meal.

FIGS. 11A and 11B show graphical representations of the effect of gOBG3(3×25 μg ip) on plasma FFA in C57BL6/J mice following a high fat meal.FIG. 11A shows FFA as mM; FIG. 11B shows FFA as a percent of thestarting value.

FIGS. 12A and 12B show graphical representations of the effect of gOBG3(3×25 μg) on plasma leptin in C57BL6/J mice following a high fat meal.FIG. 12A shows leptin as ng/mL; FIG. 12B shows leptin as a percent ofthe starting value.

FIGS. 13A and 13B show graphical representations of the effect of gOBG3(3×25 μg) on plasma Insulin in C57BL6/J mice following a high fat meal.FIG. 13A shows insulin levels in ng/mL; FIG. 13B shows insulin as apercent of the starting value.

FIGS. 14A and 14B show graphical representations of the effect of OBG3on plasma FFA in C57BL6/J mice following a high fat meal. At t=2 hours asignificant reduction in FFA was seen for both treatment groups(p<0.05). FIG. 14A shows FFA levels in mM; FIG. 14B shows FFA as apercent of the starting value.

FIGS. 15A and 15B show graphical representations of the effect of OBG3on plasma TG in C57BL6/J mice following a high fat meal. FIG. 15A showsTG levels in mg/dl; FIG. 15B shows TG as a percent of the startingvalue.

FIGS. 16A and 16B show graphical representations of the effect of OBG3on plasma glucose in C57BL6/J mice following a high fat meal. FIG. 16Ashows glucose levels as mg/dl; FIG. 16B shows glucose levels as apercent of the starting value.

FIG. 17 shows a table identifying additional APM1 SNPs. Informationconcerning Known Base Changes, Location, Prior Markers, Amplicon, andForward and Reverse primers for microsequencing are shown.

FIGS. 18A and 18B show graphical representations of the effect ofgACRP30 injection in mice on the FFA (FIG. 18A) and glucose (FIG. 18B)increases resulting from epinephrine injection.

FIG. 19 shows a graphical representation of the effect of gACRP30treatment on fatty acid metabolism in muscle isolated from mice.Treatments shown are control (yellow) and gACRP30 (red).

FIGS. 20A and 20B show a graphical representation of the effect ofgACRP30 treatment on triglyceride content of muscle and liver isolatedfrom mice.

FIGS. 21A, 21B, 21C, & 21D show graphical representations of the effectof gACRP30 treatment on weight gain & loss in mice. Treatments shown aresaline (diamond), ACRP30 (filled square), and gACRP30 (triangle). FIG.21A shows results of treatment of mice after 19 days on a high fat diet.FIG. 21B shows results of treatment of mice after 6 months on a high fatdiet.

FIG. 22 shows a table of the tested blood chemistry values with salineinjections, ACRP30 injections, or gACRP30 injections.

FIG. 23 shows a graph depicting the removal of plasma FFAs afterIntralipid injection following treatment with gACRP30 (diamonds) or asaline control (triangles).

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO:1 is the nucleotide sequence of cDNA with an open readingframe which location is indicated as featured.

SEQ ID NO:2 is the amino acid sequence of protein encoded by the cDNA ofSEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence of cDNA with an open readingframe which location is indicated as featured.

SEQ ID NO:4 is the amino acid sequence of protein encoded by the cDNA ofSEQ ID NO:3.

SEQ ID NO:5 is genomic nucleotide sequence comprising the gene encodingthe protein of SEQ ID NO:2.

The appended Sequence Listing is hereby incorporated by reference in itsentirety.

DETAILED DISCLOSURE OF THE INVENTION

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

As used interchangeably herein, the terms “oligonucleotides”, and“polynucleotides” and nucleic acid include RNA, DNA, or RNA/DNA hybridsequences of more than one nucleotide in either single chain or duplexform. The terms encompass “modified nucleotides” which comprise at leastone modification, including by way of example and not limitation: (a) analternative linking group, (b) an analogous form of purine, (c) ananalogous form of pyrimidine, or (d) an analogous sugar. For examples ofanalogous linking groups, purines, pyrimidines, and sugars see forexample PCT publication No. WO 95/04064. The polynucleotide sequences ofthe invention may be prepared by any known method, including synthetic,recombinant, ex vivo generation, or a combination thereof, as well asutilizing any purification methods known in the art.

The terms polynucleotide construct, recombinant polynucleotide andrecombinant polypeptide are used herein consistently with their use inthe art. The terms “upstream” and “downstream” are also used hereinconsistently with their use in the art. The terms “base paired” and“Watson & Crick base paired” are used interchangeably herein andconsistently with their use in the art. Similarly, the terms“complementary”, “complement thereof”, “complement”, “complementarypolynucleotide”, “complementary nucleic acid” and “complementarynucleotide sequence” are used interchangeably herein and consistentlywith their use in the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention that has been separated fromother compounds including, but not limited to, other nucleic acids,carbohydrates, lipids and proteins (such as the enzymes used in thesynthesis of the polynucleotide). Purified can also refer to theseparation of covalently closed polynucleotides from linearpolynucleotides, or vice versa, for example. A polynucleotide issubstantially pure when at least about 50%, 60%, 75%, or 90% of a samplecontains a single polynucleotide sequence. In some cases this involves adetermination between conformations (linear versus covalently closed). Asubstantially pure polynucleotide typically comprises about 50, 60, 70,80, 90, 95, 99% weight/weight of a nucleic acid sample. Polynucleotidepurity or homogeneity may be indicated by a number of means well knownin the art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polynucleotide band uponstaining the gel. For certain purposes, higher resolution can beachieved by using HPLC or other means well known in the art.

Similarly, the term “purified” is used herein to describe a polypeptideof the invention that has been separated from other compounds including,but not limited to, nucleic acids, lipids, carbohydrates and otherproteins. In some preferred embodiments, a polypeptide is substantiallypure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.5% of the polypeptide molecules of a sample have a singleamino acid sequencesequence. In some preferred embodiments, asubstantially pure polypeptide typically comprises about 50%, 60%, 70%,80%, 90% 95%, 96%, 97%, 98%, 99% or 99.5% weight/weight of a proteinsample. Polypeptide purity or homogeneity is indicated by a number ofmethods well known in the art, such as agarose or polyacrylamide gelelectrophoresis of a sample, followed by visualizing a singlepolypeptide band upon staining the gel. For certain purposes, higherresolution can be achieved by using HPLC or other methods well known inthe art.

Further, as used herein, the term “purified” does not require absolutepurity, rather, it is intended as a relative definition. Purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated. Altematively,purification may be expressed as “at least” a percent purity relative toheterologous polynucleotides (DNA, RNA or both) or polypeptides. As apreferred embodiment, the polynucleotides or polypeptides of the presentinvention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologouspolynucleotides or polypeptides. As a further preferred embodiment thepolynucleotides or polypeptides have an “at least” purity ranging fromany number, to the thousandth position, between 90% and 100% (e.g., atleast 99.995% pure) relative to heterologous polynucleotidesorpolypeptides. Additionally, purity of the polynucleotides orpolypeptides may be expressed as a percentage (as described above)relative to all materials and compounds other than the carrier solution.Each number, to the thousandth position, may be claimed as individualspecies of purity.

The term “isolated” requires that the material be removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

Specifically excluded from the definition of “isolated” are: naturallyoccurring chromosomes (e.g., chromosome spreads), artificial chromosomelibraries, genomic libraries, and cDNA libraries that exist either as anin vitro nucleic acid preparation or as a transfected/transformed hostcell preparation, wherein the host cells are either an in vitroheterogeneous preparation or plated as a heterogeneous population ofsingle colonies. Also specifically excluded are the above librarieswherein a 5′ EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%,10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acidinserts in the vector molecules. Further specifically excluded are wholecell genomic DNA or whole cell RNA preparations (including said wholecell preparations which are mechanically sheared or enzymaticallydigested). Further specifically excluded are the above whole cellpreparations as either an in vitro preparation or as a heterogeneousmixture separated by electrophoresis (including blot transfers of thesame) wherein the polynucleotide of the invention have not been furtherseparated from the heterologous polynucleotides in the electrophoresismedium (e.g., further separating by excising a single band from aheterogeneous band population in an agarose gel or nylon blot).

The term “primer” denotes a specific oligonucleotide sequence that iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by DNA polymerase, RNApolynierase, or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., PNA as defined hereinbelow) which can be used toidentify a specific polynucleotide sequence present in a sample, saidnucleic acid segment comprising a nucleotide sequence complementary tothe specific polynucleotide sequence to be identified.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer. Thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides. For example, polypeptides that include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. As used herein, the term “OBG3” refersgenerically to the murine or human OBG3, unless otherwise specified. Theterms “ACRP30” and “AdipoQ” refer specifically to the murine form ofOBG3 and the term “APM1” refers specifically to the human form of thegene.

Without being limited by theory, the compounds/polypeptides of theinvention are capable of modulating the partitioning of dietary lipidsbetween the liver and peripheral tissues, and are thus believed to treat“diseases involving the partitioning of dietary lipids between the liverand peripheral tissues.” The term “peripheral tissues” is meant toinclude muscle and adipose tissue. In preferred embodiments, thecompounds/polypeptides of the invention partition the dietary lipidstoward the muscle. In alternative preferred embodiments, the dietarylipids are partitioned toward the adipose tissue. In other preferredembodiments, the dietary lipids are partitioned toward the liver. In yetother preferred embodiments, the compounds/polypeptides of the inventionincrease or decrease the oxidation of dietary lipids, preferably freefatty acids (FFA) by the muscle. Dietary lipids include, but are notlimited to triglycerides and free fatty acids.

Preferred diseases believed to involve the partitioning of dietarylipids include obesity and obesity-related diseases and disorders suchas obesity, impaired glucose tolerance, insulin resistance,atherosclerosis, atheromatous disease, heart disease, hypertension,stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, orType II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or TypeI diabetes). Diabetes-related complications to be treated by the methodsof the invention include microangiopathic lesions, ocular lesions,retinopathy, neuropathy, and renal lesions. Heart disease includes, butis not limited to, cardiac insufficiency, coronary insufficiency, andhigh blood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother metabolic-related diseases or disorders of the invention includingcachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

The term “heterologous”, when used herein, is intended to designate anypolypeptide or polynucleotide other than an OBG3 or OBG3 polypeptide ora polynucleotide encoding a gOBG3 polypeptide of the present invention.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. A defined meaning setforth in the M.P.E.P. controls over a defined meaning in the art and adefined meaning set forth in controlling Federal Circuit case lawcontrols over a meaning set forth in the M.P.E.P. With this in mind, theterms may be substituted for one another throughout the instantapplication in order to attach the specific meaning associated with eachterm.

The term “host cell recombinant for” a particular polynucleotide of thepresent invention, means a host cell that has been altered by the handsof man to contain said polynucleotide in a way not naturally found insaid cell. For example, said host cell may be transiently or stablytransfected or transduced with said polynucleotide of the presentinvention.

The term “obesity” as used herein is defined in the WHO classificationsof weight (Kopelman (2000) Nature 404:635643). Underweight is less than18.5 (thin); Healthy is 18.5-24.9 (normal); grade 1 overweight is25.0-29.9 (overweight); grade 2 overweight is 30.0-39.0 (obesity); grade3 overweight is greater than or equal to 40.0 BMI. BMI is body massindex (morbid obesity) and is kg/m². Waist circumference can also beused to indicate a risk of metabolic complications where in men acircumference of greater than or equal to 94 cm indicates an increasedrisk, and greater than or equal to 102 cm indicates a substantiallyincreased risk. Similarly for women, greater than or equal to 88 cmindicates an increased risk, and greater than or equal to 88 cmindicates a substantially increased risk. The waist circumference ismeasured in cm at midpoint between lower border of ribs and upper borderof the pelvis. Other measures of obesity include, but are not limitedto, skinfold thickness which is a measurement in cm of skinfoldthickness using calipers, and bioimpedance, which is based on theprinciple that lean mass conducts current better than fat mass becauseit is primarily an electrolyte solution; measurement of resistance to aweak current (impedance) applied across extremities provides an estimateof body fat using an empirically derived equation.

The term “energy intake” as used herein is defined as the energyintroduced into an individual from total caloric intake, i.e., the totalenergy from food and liquid diet.

The term “energy expenditure” as used herein is defined as total energyexpenditure (TEE), which includes resting energy expenditure (REE), thethermic effect of feeding (TEF), and activities such as exercise. Both“energy intake” and “energy expenditure” are defined by Rosenbaum et al.[Am J Clin Nutr June 2000; 71(6):1421-32), which is hereby incorporatedby reference in its entirety].

The term “maintenance of weight loss” as used herein is defined assustaining a stable weight in an individual that is 10-20% below theinitial, obese weight of the individual. Preferably, the new maintainedweight after weight loss is a healthy weight (as defined herein). Whenthe maintenance of weight loss is practiced for cosmetic purposes, theindividual has a BMI of at least 20 and no more than 25. As defined forthe treatment of obesity by means of maintaining weight loss, theindividual may have a BMI of at least 20.

The term “diabetes” as used herein is intended to encompass the usualdiagnosis of diabetes made from any of the methods included, but notlimited to, the following list: symptoms of diabetes (eg. polyuria,polydipsia, polyphagia) plus casual plasma glucose levels of greaterthan or equal to 200 mg/dl, wherein casual plasma glucose is defined anytime of the day regardless of the timing of meal or drink consumption; 8hour fasting plasma glucose levels of less than or equal to 126 mg/dl;and plasma glucose levels of greater than or equal to 200 mg/dl 2 hoursfollowing oral administration of 75 g anhydrous glucose dissolved inwater.

The term “impaired glucose tolerance (IGT)” as used herein is intendedto indicate that condition associated with insulin-resistance that isintermediate between frank, NIDDM and normal glucose tolerance (NGT). Ahigh percentage of the IGT population is known to progress to NIDDMrelative to persons with normal glucose tolerance (Sad et al., New EnglJ Med 1988; 319:1500-6 which disclosure is hereby incorporated byreference in its entirety). Thus, by providing therapeutics and methodsfor reducing or preventing IGT, i.e., for normalizing insulinresistance, the progression to NIDDM can be delayed or prevented. IGT isdiagnosed by a procedure wherein an affected person's postprandialglucose response is determined to be abnormal as assessed by 2-hourpostprandial plasma glucose levels. In this test, a measured amount ofglucose is given to the patient and blood glucose levels measuredregular intervals, usually every half hour for the first two hours andevery hour thereafter. In a “normal” or non-IGT individual, glucoselevels rise during the first two hours to a level less than 140 mg/dland then drop rapidly. In an IGT individual, the blood glucose levelsare higher and the drop-off level is at a slower rate.

The term “Insulin-Resistance Syndrome” as used herein is intended toencompass the cluster of abnormalities resulting from an attempt tocompensate for insulin resistance that sets in motion a series of eventsthat play an important role in the development of both hypertension andcoronary artery disease (CAD), such as premature atheroscleroticvascular disease. Increased plasma triglyceride and decreasedHDL-cholesterol concentrations, conditions that are known to beassociated with CAD, have also been reported to be associated withinsulin resistance. Thus, by providing therapeutics and methods forreducing or preventing insulin resistance, the invention providesmethods for reducing and/or preventing the appearance ofinsulin-resistance syndrome.

The term “polycystic ovary syndrome (PCOS)” as used herein is intendedto designate that etiologically unassigned disorder of premenopausalwomen, affecting 5-10% of this population, characterized byhyperandrogenism, chronic anovulation, defects in insulin action,insulin secretion, ovarian steroidogenesis and fibrinolysis. Women withPCOS frequently are insulin resistant and at increased risk to developglucose intolerance or NIDDM in the third and fourth decades of life(Dunaif et al. (1996) J Clin Endocrinol Metab 81:3299 which disclosureis hereby incorporated by reference in its entirety). Hyperandrogenismalso is a feature of a variety of diverse insulin-resistant states, fromthe type A syndrome, through leprechaunism and lipoatrophic diabetes, tothe type B syndrome, when these conditions occur in premenopausal women.It has been suggested that hyperinsulinemia per se causeshyperandrogenism. Insulin-sensitizing agents, e.g., troglitazone, havebeen shown to be effective in PCOS and that, in particular, the defectsin insulin action, insulin secretion, ovarian steroidogenosis andfibrinolysis are improved (Ehrman et al. (1997) J Clin Invest 100:1230which disclosure is hereby incorporated by reference in its entirety),such as in insulin-resistant humans.

The term “insulin resistance” as used herein is intended to encompassthe usual diagnosis of insulin resistance made by any of a number ofmethods, such as the intravenous glucose tolerance test or measurementof the fasting insulin level. It is well known that there is anexcellent correlation between the height of the fasting insulin leveland the degree of insulin resistance. Therefore, one could use elevatedfasting insulin levels as a surrogate marker for insulin resistance forthe purpose of identifying which normal glucose tolerance (NGT)individuals have insulin resistance. Another way to do this is to followthe approach as disclosed in The New England Journal of Medicine, No. 3,pp. 1188 (1995) (which disclosure is hereby incorporated by reference inits entirety), i.e. to select obese subjects as an initial criterion forentry into the treatment group. Some obese subjects have impairedglucose tolerance (IGT) while others have normal glucose tolerance(NGT). Since essentially all obese subjects are insulin resistant, i.e.even the NGT obese subjects are insulin resistant and have fastinghyperinsulinemia. Therefore, the target of the treatment according tothe present invention can be defined as NGT individuals who are obese orwho have fasting hyperinsulinemia, or who have both.

A diagnosis of insulin resistance can also be made using the euglycemicglucose clamp test. This test involves the simultaneous administrationof a constant insulin infusion and a variable rate glucose infusion.During the test, which lasts 3-4 hours, the plasma glucose concentrationis kept constant at euglycemic levels by measuring the glucose levelevery 5-10 minutes and then adjusting the variable rate glucose infusionto keep the plasma glucose level unchanged. Under these circumstances,the rate of glucose entry into the bloodstream is equal to the overallrate of glucose disposal in the body. The difference between the rate ofglucose disposal in the basal state (no insulin infusion) and theinsulin infused state, represents insulin mediated glucose uptake. Innormal individuals, insulin causes brisk and large increase in overallbody glucose disposal, whereas in NIDDM subjects, this effect of insulinis greatly blunted, and is only 20-30% of normal. In insulin resistantsubjects with either IGT or NGT, the rate of insulin stimulated glucosedisposal is about half way between normal and NIDDM. For example, at asteady state plasma insulin concentration of about 100 μU/ml (aphysiologic level) the glucose disposal rate in normal subjects is about7 mg/kg/min. In NIDDM subjects, it is about 2.5 mg/kg/min., and inpatients with IGT (or insulin resistant subjects with NGT) it is about4-5 mg/kg/min. This is a highly reproducible and precise test, and candistinguish patients within these categories. It is also known that assubjects become more insulin resistant, the fasting insulin level rises.There is an excellent positive correlation between the height of thefasting insulin level and the magnitude of the insulin resistance asmeasured by euglycemic glucose clamp tests and, therefore, this providesthe rationale for using fasting insulin levels as a surrogate measure ofinsulin resistance.

The term “agent acting on the partitioning of dietary lipids between theliver and peripheral tissues” refers to a compound or polypeptide of theinvention that modulates the partitioning of dietary lipids between theliver and the peripheral tissues as previously described. Preferably,the agent increases or decreases the oxidation of dietary lipids,preferably free fatty acids (FFA) by the muscle. Preferably the agentdecreases or increases the body weight of individuals or is used totreat or prevent an obesity-related disease or disorder such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disease, heart disease, hypertension, stroke, Syndrome X,Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions. Heart disease includes, but is notlimited to, cardiac insufficiency, coronary insufficiency, and highblood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia.

The terms “response to an agent acting on the partitioning of dietarylipids between the liver and peripheral tissues” refer to drug efficacy,including but not limited to, ability to metabolize a compound, abilityto convert a pro-drug to an active drug, and the pharmacokinetics(absorption, distribution, elimination) and the pharmacodynamics(receptor-related) of a drug in an individual.

The terms “side effects to an agent acting on the partitioning ofdietary lipids between the liver and peripheral tissues” refer toadverse effects of therapy resulting from extensions of the principalpharmacological action of the drug or to idiosyncratic adverse reactionsresulting from an interaction of the drug with unique host factors.“Side effects to an agent acting on the partitioning of dietary lipidsbetween the liver and peripheral tissues” can include, but are notlimited to, adverse reactions such as dermatologic, hematologic orhepatologic toxicities and further includes gastric and intestinalulceration, disturbance in platelet function, renal injury, nephritis,vasomotor rhinitis with profuse watery secretions, angioneurotic edema,generalized urticaria, and bronchial asthma to laryngeal edema andbronchoconstriction, hypotension, and shock.

The term “OBG3-related diseases and disordersμ as used herein refers toany disease or disorder comprising an aberrant functioning of OBG3, orwhich could be treated or prevented by modulating OBG3 levels oractivity. “Aberrant functioning of OBG3? includes, but is not limitedto, aberrant levels of expression of OBG3 (either increased ordecreased, but preferably decreased), aberrant activity of OBG3 (eitherincreased or decreased), and aberrant interactions with ligands orbinding partners (either increased or decreased). By “aberrant” is meanta change from the type, or level of activity seen in normal cells,tissues, or patients, or seen previously in the cell, tissue, or patientprior to the onset of the illness. In preferred embodiments, theseOBG3-related diseases and disorders include obesity and theobesity-related diseases and disorders described previously.

The term “cosmetic treatments” is meant to include treatments withcompounds or polypeptides of the invention that increase or decrease thebody mass of an individual where the individual is not clinically obeseor clinically thin. Thus, these individuals have a body mass index (BMI)below the cut-off for clinical obesity (e.g. below 25 kg/m²) and abovethe cut-off for clinical thinness (e.g. above 18.5 kg/m²). In addition,these individuals are preferably healthy (e.g. do not have anobesity-related disease or disorder of the invention). “Cosmetictreatments” are also meant to encompass, in some circumstances, morelocalized increases in adipose tissue, for example, gains or lossesspecifically around the waist or hips, or around the hips and thighs,for example. These localized gains or losses of adipose tissue can beidentified by increases or decreases in waist or hip size, for example.

The term “preventing” as used herein refers to administering a compoundprior to the onset of clinical symptoms of a disease or condition so asto prevent a physical manifestation of aberrations associated withobesity or OBG3. Alternatively, the term “preventing” can also be usedto signify the reduction, or severity, of clinical symptoms associatedwith a disease or condition.

The term “treating” as used herein refers to administering a compoundafter the onset of clinical symptoms.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver (e.g. physician, nurse, nurse practitioner, etc in thecase of humans; veterinarian in the case of animals, including non-humanmammals) that an individual or animal requires or will benefit fromtreatment. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that include the knowledgethat the individual or animal is ill, or will be ill, as the result of acondition that is treatable by the compounds of the invention.

The term “perceives a need for treatment” refers to a sub-clinicaldetermination that an individual desires to reduce weight for cosmeticreasons as discussed under “cosmetic treatment” above. The term“perceives a need for treatment” in other embodiments can refer to thedecision that an owner of an animal makes for cosmetic treatment of theanimal.

The term “individual” or “patient” as used herein refers to any animal,including mammals, preferably mice, rats, other rodents, rabbits, dogs,cats, swine, cattle, sheep, horses, or primates, and most preferablyhumans. The term may specify male or female or both, or exclude male orfemale.

The term “non-human animal” refers to any non-human vertebrate,including birds and more usually mammals, preferably primates, animalssuch as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits orrodents, more preferably rats or mice. Both the terms “animal” and“mammal” expressly embrace human subjects unless preceded with the term“non-human”.

The inventors have found that a fragment of OBG3, called gOBG3, is ableto significantly reduce the postprandial response of plasma free fattyacids, glucose, and triglycerides in mice fed a high fat/sucrose meal.There was no significant effect on leptin, insulin or glucagon levels.In addition, gOBG3 was found to increase muscle free fatty acidoxidation in vitro and ex vivo. Further, gOBG3 was shown to decrease andthen to prevent an increase in weight gain in mice that had been fed ahigh fat/sucrose diet for 19 days. In mice that had been maintained onthe same high fat/sucrose diet for 6 months, gOBG3 treatment resulted ina sustained weight loss over 16 days that was significant, despite beingmaintained on the high fat/sucrose diet.

The instant invention encompasses the use of OBG3 polypeptide fragmentsin the partitioning of free fatty acid (FFA) and as an important newtool to control energy homeostasis. Of the tissues that cansignificantly remove lipids from circulation and cause FFA oxidation,muscle is quantitatively the most important. Globular OBG3 is a uniqueand novel pharmacological tool that controls body weight withoutinterfering with food intake.

PREFERRED EMBODIMENTS OF THE INVENTION

I. OBG3 Polypeptide Fragments of the Invention

OBG3 polypeptide fragments that have measurable activity in vitro and invivo have been identified. These activities include, but are not limitedto, reduction of the postprandial response of plasma free fatty acids,glucose, and triglycerides in mice fed a high fat/sucrose meal (Example8), increase in muscle free fatty acid oxidation in vitro and ex vivo(Example 12), and sustained weight loss in mice on a high fat/sucrosediet (Example 14). Other assays for OBG3 polypeptide fragment activityin vitro and in vivo are also provided (Examples 4, 7, 9, 11, 13, forexample), and equivalent assays can be designed by those of ordinaryskill in the art.

In contrast, the “intact” or “full-length” OBG3 polypeptide does nothave either the in vivo or the in vitro activities that have beenidentified for gOBG3 polypeptide fragments of the invention. In mostcases, the activities are either not present or at a minimum areundetectable over control values in the assays used. In other cases, theactivities can be measured, but are present either at extremely reducedlevels and/or require significantly more protein on a molar basiscompared with the gOBG3 polypeptide fragments of the invention (see,e.g. Example 10). By “intact” or “full-length” OBG3 polypeptide as usedherein is meant the full-length polypeptide sequence of any OBG3polypeptide, from the N-terminal methionine to the C-terminal stopcodon. Examples of intact or full-length OBG3 polypeptides are found inSEQ ID NO:2 (human) and SEQ ID NO:4 (mouse). The term “OBG3 polypeptidefragments” as used herein refers to fragments of the “intact” or“full-length ” OBG3 polypeptide that have “obesity-related activity” or“insulin-like activity”. The term “gOBG3 polypeptide fragments” refersto polypeptide fragments comprised of the globular domain and is thus anarrower term than “OBG3 polypeptide fragments”. The term “fragment”means a polypeptide having a sequence that is entirely the same as part,but not all, of an intact or full-length OBG3 polypeptide. Suchfragments may be “free-standing” (i.e. not part of or fused to otherpolypeptides), or one or more fragments may be present in a singlepolypeptide. gOBG3 fragments are contiguous fragments of the full-lengthOBG3 polypeptide unless otherwise specified.

The term “obesity-related activity” as used herein refers to at leastone, and preferably all, of the activities described herein for OBG3polypeptide fragments. Assays for the determination of these activitiesare provided herein (e.g. Examples 4, 7-9, 11-14), and equivalent assayscan be designed by those with ordinary skill in the art. Optionally,“obesity-related activity” can be selected from the group consisting oflipid partitioning, lipid metabolism, and insulin-like activity, or anactivity within one of these categories. By “lipid partitioning”activity is meant the ability to effect the location of dietary lipidsamong the major tissue groups including, adipose tissue, liver, andmuscle. The inventors have shown that OBG3 polypeptide fragments of theinvention play a role in the partitioning of lipids to the muscle, liveror adipose tissue. By “lipid metabolism” activity is meant the abilityto influence the metabolism of lipids. The inventors have shown thatOBG3 polypeptide fragments of the invention have the ability to affectthe level of free fatty acids in the plasma as well as to increase themetabolism of lipids in the muscle through free fatty acid oxidationexperiments (Examples 4, 8, 10, 11, 12) and to transiently affect thelevels of triglycerides in the plasma and the muscle (Examples 8, 1013). By “insulin-like” activity is meant the ability of OBG3 polypeptidefragments to modulate the levels of glucose in the plasma. The inventorshave found that OBG3 polypeptide fragments do not significantly impactinsulin levels but do impact glucose levels similarly to the effects ofinsulin (Examples 9 & 10). These effects are not seen in the presence ofmultimers of gOBG3 polypeptide fragment homotrimer or are significantlygreater in the presence of non-multimeric gOBG3 polypeptide fragmenttrimer compared with multimers of gOBG3 polypeptide fragment homotrimer.

The term “significantly greater” as used herein refers to a comparisonof the activity of an non-multimeric gOBG3 polypeptide fragmenthomotrimer in an obesity-related assay compared with the activity ofmultimers of gOBG3 polypeptide fragment homotrimer in the same assay. By“significantly” as used herein is meant statistically significant as itis typically determined by those with ordinary skill in the art. Forexample, data are typically calculated as a mean i SEM, and ap-value≦0.05 is considered statistically significant. Statisticalanalysis is typically done using either the unpaired Student's t test orthe paired Student's t test, as appropriate in each study. Examples of asignificant change in activity as a result of the presence of an OBG3polypeptide fragment of the invention compared to the presence of afull-length OBG3 polypeptide include an increase or a decrease in agiven parameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75%. One or more, but not necessarily all,of the measurable parameters will change significantly in the presenceof OBG3 polypeptide fragments as compared to in the presence of anintact OBG3 polypeptide.

Representative “obesity-related assays” are provided in Examples 4, 7-9,and 11-14. These assays include, but are not limited to, methods ofmeasuring the postprandial response, methods of measuring free fattyacid oxidation, and methods of measuring weight modulation. In preferredembodiments, the post-prandial response is measured in non-humananimals, preferably mice. In preferred embodiments changes in dietarylipids are measured, preferably free fatty acids and/or triglycerides.In other embodiments, other physiologic parameters are measuredincluding, but not limited to, levels of glucose, insulin, and leptin.In other preferred embodiments, free fatty acid oxidation is measured incells in vitro or ex vivo, preferably in muscle cells or tissue ofnon-human animals, preferably mice. In yet other preferred embodimentsweight modulation is measured in human or non-human animals, preferablyrodents (rats or mice), primates, canines, felines or procines on a highfat/sucrose diet. Optionally, “obesity-related activity” includes otheractivities not specifically identified herein. In general, “measurableparameters” relating to obesity and the field of metabolic research canbe selected from the group consisting of free fatty acid levels, freefatty acid oxidation, triglyceride levels, glucose levels, insulinlevels, leptin levels, food intake, weight, leptin and lipoproteinbinding, uptake and degradation and lipolysis stimulated receptor (LSR)expression.

In these obesity-related assays, preferred OBG3 polypeptide fragments ofthe invention, but not full-length OBG3 polypeptides, would cause asignificant change in at least one of the measurable parameters selectedfrom the group consisting of post-prandial lipemia, free fatty acidlevels, triglyceride levels, glucose levels, free fatty acid oxidation,and weight. Alternatively, preferred OBG3 polypeptide fragments of theinvention, but not full-length OBG3 polypeptides, would have asignificant change in at least one of the measurable parameters selectedfrom the group consisting of an increase in LSR activity, an increase inleptin activity and an increase in lipoprotein activity. By “LSR”activity is meant expression of LSR on the surface of the cell, or in aparticular conformation, as well as its ability to bind, uptake, anddegrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor.

The invention is drawn, inter alia, to isolated, purified or recombinantOBG3 polypeptide fragments. OBG3 polypeptide fragments of the inventionare useful for reducing or increasing (using antagonists of OBG3polypeptides) body weight either as a cosmetic treatment or fortreatment or prevention of obesity-related diseases and disorders. OBG3polypeptide fragments are also useful inter alia in screening assays foragonists or antagonists of OBG3 fragment activity; for raising OBG3polypeptide fragment-specific antibodies; and in diagnostic assays. Whenused for cosmetic treatments, or for the treatment or prevention ofobesity-related diseases, disorders, or conditions, one or more OBG3polypeptide fragments can be provided to a subject. Thus, variousfragments of the full-length protein can be combined into a “cocktail”for use in the various treatment regimens.

The full-length OBG3 polypeptide is comprised of at least four distinctregions including:

-   1. an N-terminal putative signal sequence about from amino acids    1-17 of SEQ ID NO:2 or SEQ ID NO:4;-   2. an N-terminally disposed unique region about from amino acids    18-41 of SEQ ID NO:2 or 18-44 of SEQ ID NO:4;

3. a collagen-like region about from amino acids 42-107 of SEQ ID NO:2or 45-110 of SEQ ID NO:4; and

-   4. a globular domain about from amino acids 108-244 of SEQ ID NO:2    or 111-247 of SEQ ID NO:4.

The term “collagen residues” is used in the manner standard in the artto mean the amino acid triplet glycine, X, Y, where X and Y can be anyamino acid.

The OBG3 polypeptide fragments of the present invention are preferablyprovided in an isolated form, and may be partially or substantiallypurified. A recombinantly produced version of an OBG3 polypeptidefragment can be substantially purified by the one-step method describedby Smith et al. ((1988) Gene 67(1):3140) or by the methods describedherein or known in the art (see, e.g., Examples 1-3). Fragments of theinvention also can be purified from natural or recombinant sources usingantibodies directed against the polypeptide fragments of the inventionby methods known in the art of protein purification.

Preparations of OBG3 polypeptide fragments of the invention involving apartial purification of or selection for the OBG3 polypeptide fragmentsare also specifically contemplated. These crude preparations areenvisioned to be the result of the concentration of cells expressingOBG3 polypeptide fragments with perhaps a few additional purificationsteps, but prior to complete purification of the fragment. The cellsexpressing OBG3 polypeptide fragments are present in a pellet, they arelysed, or the crude polypeptide is lyophilized, for example.

gOBG3 polypeptide fragments, and polynucleotides encoding the same, canbe any integer in length from 6 consecutive amino acids to 1 amino acidless than a full-length OBG3 polypeptide. Thus, for human OBG3 of SEQ IDNO:2, a gOBG3 polypeptide fragment can be any integer of consecutiveamino acids from 6 to 243; for mouse OBG3 of SEQ ID NO:4, a gOBG3polypeptide fragment can be any integer of consecutive amino acids from6 to 246, for example. The term “integer” is used herein in itsmathematical sense and thus representative integers include: 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245 and 246.

Each OBG3 fragment as described above can be further specified in termsof its N-terminal and C-terminal positions. For example, everycombination of a N-terminal and C-terminal position that fragments offrom 6 contiguous amino acids to 1 amino acid less than the full-lengthOBG3 polypeptide could occupy, on any given intact and contiguousfull-length OBG3 polypeptide sequence are included in the presentinvention. Thus, a 6 consecutive amino acid fragment could occupypositions selected from the group consisting of 1-6, 2-7, 3-8, 4-9,5-10, 6-11, 7-12, 8-13, 9-14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20,16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-28, 24-29, 25-30,26-31, 27-32, 28-33, 29-34, 30-35, 31-36, 32-37, 33-38, 34-39, 35-40,36-41, 37-42, 38-43, 39-44, 40-45, 41-46, 42-47, 43-48, 44-49, 45-50,46-51, 47-52, 48-53, 49-54, 50-55, 51-56, 52-57, 53-58, 54-59, 55-60,56-61, 57-62, 58-63, 59-64, 60-65, 61-66, 62-67, 63-68, 64-69, 65-70,66-71, 67-72, 68-73, 69-74, 70-75, 71-76, 72-77, 73-78, 74-79, 75-80,76-81, 77-82, 78-83, 79-84, 80-85, 81-86, 82-87, 83-88, 84-89, 85-90,86-91, 87-92, 88-93, 89-94, 90-95, 91-96, 92-97, 93-98, 94-99, 95-100,96-101, 97-102, 98-103, 99-104, 100-105, 101-106, 102-107, 103-108,104-109, 105-110, 106-111, 107-112, 108-113, 109-114, 110-115, 111-116,112-117, 113-118, 114-119, 115-120, 116-121, 117-122, 118-123, 119-124,120-125, 121-126, 122-127, 123-128, 124-129, 125-130, 126-131, 127-132,128-133, 129-134, 130-135, 131-136, 132-137, 133-138, 134-139, 135-140,136-141, 137-142, 138-143, 139-144, 140-145, 141-146, 142-147, 143-148,144-149, 145-150, 146-151, 147-152, 148-153, 149-154, 150-155, 151-156,152-157, 153-158, 154-159, 155-160, 156-161, 157-162, 158-163, 159-164,160-165, 161-166, 162-167, 163-168, 164-169, 165-170, 166-171, 167-172,168-173, 169-174, 170-175, 171-176, 172-177, 173-178, 174-179, 175-180,176-181, 177-182, 178-183, 179-184, 180-185, 181-186, 182-187, 183-188,184-189, 185-190, 186-191, 187-192, 188-193, 189-194, 190-195, 191-196,192-197, 193-198, 194-199, 195-200, 196-201, 197-202, 198-203, 199-204,200-205, 201-206, 202-207, 203-208, 204-209, 205-210, 206-211, 207-212,208-213, 209-214, 210-215, 211-216, 212-217, 213-218, 214-219, 215-220,216-221, 217-222, 218-223, 219-224, 220-225, 221-226, 222-227, 223-228,224-229, 225-230, 226-231, 227-232, 228-233, 229-234, 230-235, 231-236,232-237, 233-238, 234-239, 235-240, 236-241, 237-242, 238-243, and239-244 of SEQ ID NO:2.

Further preferred polypeptide fragments of SEQ ID NO:2, andpolynucleotides encoding the same, are selected from the groupconsisting of fragments comprising any 50 consecutive amino acidsnumbered from 1-50, 2-51, 3-52, 4-53, 5-54, 6-55, 7-56, 8-57, 9-58,10-59, 11-60, 12-61, 13-62, 14-63, 15-64, 16-65, 17-66, 18-67, 19-68,20-69, 21-70, 22-71, 23-72, 24-73, 25-74, 26-75, 27-76, 28-77, 29-78,30-79, 31-80, 32-81, 33-82, 34-83, 35-84, 36-85, 37-86, 38-87, 39-88,40-89, 41-90, 42-91, 43-92, 44-93, 45-94, 46-95, 47-96, 48-97, 49-98,50-99, 51-100, 52-101, 53-102, 54-103, 55-104, 56-105, 57-106, 58-107,59-108, 60-109, 61-110, 62-111, 63-112, 64-113, 65-114, 66-115, 67-116,68-117, 69-118, 70-119, 71-120, 72-121, 73-122, 74-123, 75-124, 76-125,77-126, 78-127, 79-128, 80-129, 81-130, 82-131, 83-132, 84-133, 85-134,86-135, 87-136, 88-137, 89-138, 90-139, 91-140, 92-141, 93-142, 94-143,95-144, 96-145, 97-146, 98-147, 99-148, 100-149, 101-150, 102-151,103-152, 104-153, 105-154, 106-155, 107-156, 108-157, 109-158, 110-159,111-160, 112-161, 113-162, 114-163, 115-164, 116-165, 117-166, 118-167,119-168, 120-169, 121-170, 122-171, 123-172, 124-173, 125-174, 126-175,127-176, 128-177, 129-178, 130-179, 131-180, 132-181, 133-182, 134-183,135-184, 136-185, 137-186, 138-187, 139-188, 140-189, 141-190, 142-191,143-192, 144-193, 145-194, 146-195, 147-196, 148-197, 149-198, 150-199,151-200, 152-201, 153-202, 154-203, 155-204, 156-205, 157-206, 158-207,159-208, 160-209, 161-210, 162-211, 163-212, 164-213, 165-214, 166-215,167-216, 168-217, 169-218, 170-219, 171-220, 172-221, 173-222, 174-223,175-224, 176-225, 177-226, 178-227, 179-228, 180-229, 181-230, 182-231,183-232, 184-233, 185-234, 186-235, 187-236, 188-237, 189-238, 190-239,191-240, 192-241, 193-242, 194-243, 195-244 of SEQ ID NO:2.

Further preferred polypeptide fragments of SEQ ID NO:2, andpolynucleotides encoding the same, are selected from the groupconsisting of fragments comprising any 100 consecutive amino acidsnumbered from 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107,9-108, 10-109, 11-110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116,18-117, 19-118, 20-119, 21-120, 22-121, 23-122, 24-123, 25-124, 26-125,27-126, 28-127, 29-128, 30-129, 31-130, 32-131, 33-132, 34-133, 35-134,36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143,45-144, 46-145, 47-146, 48-147, 49-148, 50-149, 51-150, 52-151, 53-152,54-153, 55-154, 56-155, 57-156, 58-157, 59-158, 60-159, 61-160, 62-161,63-162, 64-163, 65-164, 66-165, 67-166, 68-167, 69-168, 70-169, 71-170,72-171, 73-172, 74-173, 75-174, 76-175, 77-176, 78-177, 79-178, 80-179,81-180, 82-181, 83-182, 84-183, 85-184, 86-185, 87-186, 88-187, 89-188,90-189, 91-190, 92-191, 93-192, 94-193, 95-194, 96-195, 97-196, 98-197,99-198, 100-199, 101-200, 102-201, 103-202, 104-203, 105-204, 106-205,107-206, 108-207, 109-208, 110-209, 111-210, 112-211, 113-212, 114-213,115-214, 116-215, 117-216, 118-217, 119-218, 120-219, 121-220, 122-221,123-222, 124-223, 125-224, 126-225, 127-226, 128-227, 129-228, 130-229,131-230, 132-231, 133-232, 134-233, 135-234, 136-235, 137-236, 138-237,139-238, 140-239, 141-240, 142-241, 143-242, 144-243, and 145-244.

A 238 consecutive amino acid fragment could occupy positions selectedfrom the group consisting of 1-238, 2-239, 3-240, 4-241, 5-242, 6-243and 7-244 of SEQ ID NO:2. Similarly, the positions occupied by all theother fragments of sizes between 6 amino acids and 243 amino acids onSEQ ID NO:2 are included in the present invention and can also beimmediately envisaged based on the examples for fragments of 6, 50, 100or 238 consecutive amino acids listed above, and therefore, are notindividually listed solely for the purpose of not unnecessarilylengthening the specification. Furthermore, the positions occupied byfragments of 6 to 246 consecutive amino acids on SEQ ID NO:4 areincluded in the present invention and can also be immediately envisagedbased on these two examples and therefore are not individually listedsolely for the purpose of not unnecessarily lengthening thespecification. In preferred embodiments, gOBG3 polypeptide fragments,and polynucleotides encoding the same, having unexpected activity areselected from amino acids numbered from 18-244, 19-244, 20-244, 21-244,22-244, 23-244, 24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244,31-244, 32-244, 33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244,40-244, 41-244, or 42-244 of SEQ ID NO:2 wherein the cysteine atposition 36 is replaced by said substitute amino acid. In preferredembodiments, gOBG3 polypeptide fragments, and polynucleotides encodingthe same, having unexpected activity are selected from amino acidsnumbered from 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247,25-247, 26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247,34-247, 35-247,36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247,43-247, 44-247, or 45-247 of SEQ ID NO:4 wherein the cysteine atposition 39 is replaced by said substitute amino acid. In otherpreferred embodiments, gOBG3 polypeptide fragments, and polynucleotidesencoding the same, having unexpected activity are selected from aminoacids numbered from 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 ofSEQ ID NO:2. In other preferred embodiments, gOBG3 polypeptidefragments, and polynucleotides encoding the same, having unexpectedactivity are selected from amino acids numbered from 40-247, 41-247,42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4.

The gOBG3 polypeptide fragments of the present invention mayalternatively be described by the formula “n to c” (inclusive); where“n” equals the N-terminal most amino acid position (as defined by thesequence listing) and “c” equals the C-terminal most amino acid position(as defined by the sequence listing) of the polypeptide; and furtherwhere “n” equals an integer between 1 and the number of amino acids ofthe full length polypeptide sequence of the present invention minus 5(239 for SEQ ID NO:2 and 242 for SEQ ID NO:4); and where “c” equals aninteger between 6 and the number of amino acids of the full-lengthpolypeptide sequence (244 for SEQ ID NO:2 and 247 for SEQ ID NO:4); andwhere “n” is an integer smaller then “c” by at least 6. Therefore, forSEQ ID NO:2, “n” is any integer selected from the list consisting of: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235,236, 237, 238, and 239; and “c” is any integer selected from the groupconsisting of: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244. Every combination of “n”and “c” positions are included as specific embodiments of the invention.Moreover, the formula “n” to “c” may be modified as ‘“n1-n2” to“c1-c2”’, wherein “n1-n2” and “c1-c2” represent positional rangesselected from any two integers above which represent amino acidpositions of the sequence listing. Alternative formulas include ‘“n1-n2”to “c”’ and ‘“n” to “c1-c2”’. In preferred embodiment, gOBG3 polypeptidefragments of the invention may be described by the formula where n1=18,n2=42, and c=244 of SEQ ID NO:2 wherein the cysteine at position 36 isreplaced by an amino acid other than cysteine, preferably serine, or bythe formula n1=18, n2=45, and c=247 of SEQ ID NO:4 wherein the cysteineat position 39 is replaced by an amino acid other than cysteine,preferably serine. In other preferred embodiment, gOBG3 polypeptidefragments of the invention may be described by the formula where n1=37,n2=42, and c=244 of SEQ ID NO:2 or by the formula n1=40, n2=45, andc=247 of SEQ ID NO:4.

These specific embodiments, and other polypeptide and polynucleotidefragment embodiments described herein may be modified as being “atleast”, “equal to”, “equal to or less than”, “less than”, “at least______ but not greater than ______” or “from ______ to ______” aspecified size or specified N-terminal and/or C-terminal positions. Itis noted that all ranges used to describe any embodiment of the presentinvention are inclusive unless specifically set forth otherwise.

The present invention also provides for the exclusion of any individualfragment specified by N-terminal and C-terminal positions or of anyfragment specified by size in amino acid residues as described above. Inaddition, any number of fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above may beexcluded as individual species. Further, any number of fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above may make up a polypeptide fragment inany combination and may optionally include non-OBG3 polypeptide sequenceas well.

gOBG3 polypeptide fragments of the invention include variants,fragments, analogs and derivatives of the gOBG3 polypeptide fragmentsdescribed above, including modified gOBG3 polypeptide fragments.

Also preferred are proteolytically cleaved fragments of OBG3 polypeptideof SEQ ID NO:2 or SEQ ID NO:4. Particularly preferred is OBG3polypeptide fragment of amino acids 85-244 of SEQ ID NO:2 made bycollagenase cleavage of OBG3 polypeptide of SEQ ID NO:2. Particularlypreferred is OBG3 polypeptide fragment of amino acids 88-247 of SEQ IDNO:4 made by collagenase cleavage of OBG3 polypeptide of SEQ ID NO:4.Particularly preferred is OBG3 polypeptide fragment of amino acids85-244 of OBG3 polypeptide of SEQ ID NO:2 made by matrixmetalloproteinase-1 (MMP-1) cleavage of SEQ ID NO:2. Particularlypreferred is OBG3 polypeptide fragment of amino acids 88-247 of SEQ IDNO:4 made by matrix metalloproteinase-1 (MMP-1) cleavage of OBG3polypeptide of SEQ ID NO:4. Particularly preferred is OBG3 polypeptidefragment of amino acids 34-244 of SEQ ID NO:2 wherein the cysteine atposition 36 is replaced by an amino acid other than cysteine, preferablyserine, made by plasmin cleavage of OBG3 polypeptide of SEQ ID NO:2wherein the cysteine at position 36 is replaced by said amino acid otherthan cysteine, preferably serine. Particularly preferred is OBG3polypeptide fragment of amino acids 37-247 of SEQ ID NO:4 wherein thecysteine at position 39 is replaced by an amino acid other thancysteine, preferably serine, made by plasmin cleavage of OBG3polypeptide of SEQ ID NO:4 wherein the cysteine at position 36 isreplaced by said amino acid other than cysteine, preferably serine. Alsoparticularly preferred is OBG3 polypeptide fragment of amino acids34-244 of SEQ ID NO:2 made by plasmin cleavage of OBG3 polypeptide ofSEQ ID NO:2. Also particularly preferred is OBG3 polypeptide fragment ofamino acids 37-244 of SEQ ID NO:4 made by plasmin cleavage of OBG3polypeptide of SEQ ID NO:4. Particularly preferred is OBG3 polypeptidefragment of amino acids 34-244 of SEQ ID NO:2 wherein the cysteine atposition 36 is replaced by an amino acid other than cysteine, preferablyserine, made by trypsin cleavage of OBG3 polypeptide of SEQ ID NO:2wherein the cysteine at position 36 is replaced by said amino acid otherthan cysteine, preferably serine. Particularly preferred is OBG3polypeptide fragment of amino acids 37-247 of SEQ ID NO:4 wherein thecysteine at position 39 is replaced by an amino acid other thancysteine, preferably serine, made by trypsin cleavage of OBG3polypeptide of SEQ ID NO:4 wherein the cysteine at position 36 isreplaced by said amino acid other than cysteine, preferably serine. Alsoparticularly preferred is OBG3 polypeptide fragment of amino acids34-244 of SEQ ID NO:2 made by trypsin cleavage of OBG3 polypeptide ofSEQ ID NO:2. Also particularly preferred is OBG3 polypeptide fragment ofamino acids 37-244 of SEQ ID NO:4 made by trypsin cleavage of OBG3polypeptide of SEQ ID NO:4.

Variants

It will be recognized by one of ordinary skill in the art that someamino acids of the gOBG3 fragment sequences of the present invention canbe varied without significant effect on the structure or function of theprotein; there will be critical amino acids in the fragment sequencethat determine activity. Thus, the invention further includes variantsof gOBG3 polypeptide fragments that have obesity-related activity asdescribed above. Such variants include OBG3 fragment sequences with oneor more amino acid deletions, insertions, inversions, repeats, andsubstitutions either from natural mutations or human manipulationselected according to general rules known in the art so as to havelittle effect on activity. Guidance concerning how to makephenotypically silent amino acid substitutions is provided below.

There are two main approaches for studying the tolerance of an aminoacid sequence to change (see, Bowie, et al. (1990) Science,247,1306-10). The first method relies on the process of evolution, in whichmutations are either accepted or rejected by natural selection. Thesecond approach uses genetic engineering to introduce amino acid changesat specific positions of a cloned gene and selections or screens toidentify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions and indicate which amino acid changes arelikely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein.

Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu and Phe;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe, Tyr. In addition, the following groups ofamino acids generally represent equivalent changes: (1) Ala, Pro, Gly,Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu,Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.

Similarly, amino acids in the gOBG3 polypeptide fragment sequences ofthe invention that are essential for function can also be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham, et al. (1989)Science 244(4908):1081-5). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for obesity-related activity using assays asdescribed above. Of special interest are substitutions of charged aminoacids with other charged or neutral amino acids that may produceproteins with highly desirable improved characteristics, such as lessaggregation. Aggregation may not only reduce activity but also beproblematic when preparing pharmaceutical or physiologically acceptableformulations, because aggregates can be immunogenic (see, e.g.,Pinckard, et al., (1967) Clin. Exp. Immunol 2:331-340; Robbins, et al.,(1987) Diabetes July; 36(7):83841; and Cleland, et al., (1993) Crit RevTher Drug Carrier Syst. 10(4):307-77).

Thus, the fragment, derivative, analog, or homolog of the gOBG3 fragmentof the present invention may be, for example: (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code (i.e. may be a non-naturally occurring aminoacid); or (ii) one in which one or more of the amino acid residuesincludes a substituent group; or (iii) one in which the gOBG3 fragmentis fused with another compound, such as a compound to increase thehalf-life of the fragment (for example, polyethylene glycol); or (iv)one in which the additional amino acids are fused to the above form ofthe fragment, such as an IgG Fc fusion region peptide or leader orsecretory sequence or a sequence which is employed for purification ofthe above form of the fragment or a pro-protein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a gOBG3 polypeptide fragment havingan amino acid sequence which contains at least one conservative aminoacid substitution, but not more than 50 conservative amino acidsubstitutions, not more than 40 conservative amino acid substitutions,not more than 30 conservative amino acid substitutions, and not morethan 20 conservative amino acid substitutions. Also provided arepolypeptides which comprise the amino acid sequence of a gOBG3 fragment,having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

Another specific embodiment of a modified gOBG3 fragment of theinvention is a polypeptide that is resistant to proteolysis, for examplea gOBG3 fragment in which a —CONH— peptide bond is modified and replacedby one or more of the following: a (CH2NH) reduced bond; a (NHCO) retroinverso bond; a (CH2-O) methylene-oxy bond; a (CH2-S) thiomethylenebond; a (CH2CH2) carba bond; a (CO—CH2) cetomethylene bond; a (CHOH—CH2)hydroxyethylene bond); a (N—N) bound; a E-alcene bond; or a —CH═CH—bond. Thus, the invention also encompasses a gOBG3 fragment or a variantthereof in which at least one peptide bond has been modified asdescribed above.

In addition, amino acids have chirality within the body of either L orD. In some embodiments it is preferable to alter the chirality of theamino acids in the gOBG3 polypeptide fragments of the invention in orderto extend half-life within the body. Thus, in some embodiments, one ormore of the amino acids are preferably in the L configuration. In otherembodiments, one or more of the amino acids are preferably in the Dconfiguration.

Percent Identity

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a gOBG3 fragment asdescribed above. By a polypeptide having an amino acid sequence atleast, for example, 95% “identical” to a gOBG3 fragment amino acidsequence is meant that the amino acid sequence is identical to the gOBG3polypeptide fragment sequence except that it may include up to fiveamino acid alterations per each 100 amino acids of the gOBG3 polypeptidefragment amino acid sequence. The reference sequence is the gOBG3polypeptide fragment with a sequence corresponding to the sequence ofthe sequence listing. Thus, to obtain a polypeptide having an amino acidsequence at least 95% identical to a gOBG3 fragment amino acid sequence,up to 5% (5 of 100) of the amino acid residues in the sequence may beinserted, deleted, or substituted with another amino acid compared withthe gOBG3 polypeptide fragment sequence. These alterations may occur atthe amino or carboxy tennini or anywhere between those terminalpositions, interspersed either individually among residues in thesequence or in one or more contiguous groups within the sequence.

As a practical matter, whether any particular polypeptide is apercentage identical to a gOBG3 fragment can be determinedconventionally using known computer programs. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl AcadSci USA 85(8):2444-8; Altschul et al., (1990) J Mol Biol 215(3):403-410;Thorpson et al., (1994) Nucleic Acids Res 22(2):4673-4680; Higgins etal., (1996) Meth Enzymol 266:383402; Altschul et al., (1997) Nuc AcidsRes 25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272). Ina particularly preferred embodiment, protein and nucleic acid sequencehomologies are evaluated using the Basic Local Alignment Search Tool(“BLAST”), which is well known in the art (See, e.g., Karlin andAltschul (1990) Proc Natl Acad Sci USA 87(6):2264-8; Altschul et al.,1990, 1993, 1997, all supra). In particular, five specific BLASTprograms are used to perform the following tasks:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (see, Gonnetet al., (1992) Science 256(5062):1443-5; Henikoff and Henikoff (1993)Proteins 17(1):49-61). Less preferably, the PAM or PAM250 matrices mayalso be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). TheBLAST programs evaluate the statistical significance of all high-scoringsegment pairs identified, and preferably selects those segments whichsatisfy a user-specified threshold of significance, such as auser-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (See, e.g., Karlin andAltschul, (1990) Proc Natl Acad Sci USA 87(6):2264-8). The BLASTprograms may be used with the default parameters or with modifiedparameters provided by the user. Preferably, the parameters are defaultparameters.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (1990) Comp. App. Biosci. 6:237-245. In a sequencealignment the query and subject sequences are both amino acid sequences.The result of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB amino acid alignment are:Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group=25 Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=247 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N— orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected because the FASTDB programdoes not account for N— and C-terminal truncations of the subjectsequence when calculating global percent identity. For subject sequencestruncated at the N— and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N— and C-terminal of the subject sequence,that are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what is used for the purposes of the present invention. Onlyresidues to the N— and C-termini of the subject sequence, which are notmatched/aligned with the query sequence, are considered for the purposesof manually adjusting the percent identity score. That is, only queryamino acid residues outside the farthest N— and C-terminal residues ofthe subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100-residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N— and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.

In another example, a 90-residue subject sequence is compared with a100-residue query sequence. This time the deletions are internal sothere are no residues at the N— or C-termini of the subject sequence,which are not matched/aligned with the query. In this case, the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N— and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected. No othermanual corrections are made for the purposes of the present invention.

Production

Note, throughout the disclosure, wherever OBG3 polypeptide fragments arediscussed, gOBG3fragments are specifically intended to be included as apreferred subset of OBG3 polypeptide fragments.

OBG3 polypeptide fragments are preferably isolated from human ormammalian tissue samples or expressed from human or mammalian genes inhuman or mammalian cells. The OBG3 polypeptide fragments of theinvention can be made using routine expression methods known in the art.The polynucleotide encoding the desired polypeptide fragments is ligatedinto an expression vector suitable for any convenient host. Botheukaryotic and prokaryotic host systems are used in forming recombinantpolypeptide fragments. The polypeptide fragment is then isolated fromlysed cells or from the culture medium and purified to the extent neededfor its intended use. Purification is by any technique known in the art,for example, differential extraction, salt fractionation,chromatography, centrifugation, and the like. See, for example, Methodsin Enzymology for a variety of methods for purifying proteins. Also, seeExamples 1-3 for methods previously used for OBG3 polypeptide fragments.

In an alternative embodiment, the polypeptides of the invention areisolated from milk. The polypeptides can be purified as full-length OBG3polypeptides, which can then be cleaved, if appropriate, in vitro togenerate an OBG3 fragment, or, alternatively, OBG3 fragments themselvescan be purified from the milk. Any of a large number of methods can beused to purify the present polypeptides from milk, including thosetaught in Protein Purification Applications, A Practical Approach (NewEdition), Edited by Simon Roe, AEA Technology Products and Systems,Biosciences, Harwell; Clark (1998) J Mammary Gland Biol Neoplasia3:337-50; Wilkins and Velander (1992) 49:333-8; U.S. Patent Nos.6,140,552; 6,025,540; Hennighausen, Protein Expression and Purification,vol. 1, pp. 3-8 (1990); Harris et al. (1997) Bioseparation 7:31-7;Degener et al. (1998) J Chromatog 799:125-37; Wilkins (1993) J CellBiochem Suppl. 0 (17 part A):39; the entire disclosures of each of whichare herein incorporated by reference. In a typical embodiment, milk iscentrifuged, e.g. at a relatively low speed, to separate the lipidfraction, and the aqueous supernatant is then centrifuged at a higherspeed to separate the casein in the milk from the remaining, “whey”fraction. Often, biomedical proteins are found in this whey fraction,and can be isolated from this fraction using standard chromatographic orother procedures commonly used for protein purification, e.g. asdescribed elsewhere in the present application. In one preferredembodiment, OBG3 polypeptides are purified using antibodies specific toOBG3 polypeptides, e.g. using affinity chromatography. In addition,methods can be used to isolate particular OBG3 fragments, e.g.electrophoretic or other methods for isolating proteins of a particularsize. The OBG3 polypeptides isolating using these methods can benaturally occurring, as OBG3 polypeptides have been discovered to benaturally present in the milk of mammals (see, e.g. Example 17), or canbe the result of the recombinant production of the protein in themammary glands of a non-human mammal, as described infra. In one suchembodiment, the OBG3 fragment is produced as a fusion protein with aheterologous, antigenic polypeptide sequence, which antigenic sequencecan be used to purify the protein, e.g., using standard immuno-affinitymethodology.

In addition, shorter protein fragments may be produced by chemicalsynthesis. Alternatively, the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

Any OBG3 fragment cDNA, including that in FIG. 4, can be used to expressOBG3 polypeptide fragments. The nucleic acid encoding the OBG3 fragmentto be expressed is operably linked to a promoter in an expression vectorusing conventional cloning technology. The OBG3 fragment cDNA insert inthe expression vector may comprise the coding sequence for: thefull-length OBG3 polypeptide (to be later modified); from 6 amino acidsto 1 amino acid less than the full-length OBG3 polypeptide; a gOBG3fragment; or variants and % similar polypeptides.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art, some of which aredescribed herein, and examples of which are given in the Examples(Examples 1-3). Commercially available vectors and expression systemsare available from a variety of suppliers including Genetics Institute(Cambridge, Mass.), Stratagene (La Jolla, Calif.), Promega (Madison,Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhanceexpression and facilitate proper protein folding, the codon context andcodon pairing of the sequence can be optimized for the particularexpression organism into which the expression vector is introduced, asexplained by Hatfield, et al., U.S. Pat. No. 5,082,767, the disclosuresof which are incorporated by reference herein in their entirety.

If the nucleic acid encoding OBG3 polypeptide fragments lacks amethionine to serve as the initiation site, an initiating methionine canbe introduced next to the first codon of the nucleic acid usingconventional techniques. Similarly, if the insert from the OBG3polypeptide fragment cDNA lacks a poly A signal, this sequence can beadded to the construct by, for example, splicing out the Poly A signalfrom pSG5 (Stratagene) using BglI and SalI restriction endonucleaseenzymes and incorporating it into the mammalian expression vector pXT1(Stratagene). pXT1 contains the LTRs and a portion of the gag gene fromMoloney Murine Leukemia Virus. The position of the LTRs in the constructallow efficient stable transfection. The vector includes the HerpesSimplex Thymidine Kinase promoter and the selectable neomycin gene.

The nucleic acid encoding an OBG3 fragment can be obtained by PCR from avector containing the OBG3 nucleotide sequence using oligonucleotideprimers complementary to the desired OBG3 cDNA and containingrestriction endonuclease sequences for Pst I incorporated into the 5′primer and BglII at the 5′ end of the corresponding cDNA 3′ primer,taking care to ensure that the sequence encoding the OBG3 fragment ispositioned properly with respect to the poly A signal. The purifiedfragment obtained from the resulting PCR reaction is digested with PstI,blunt ended with an exonuclease, digested with BglII, purified andligated to pXT1, now containing a poly A signal and digested with BglII.Alternative methods are presented in Examples 1-3.

Transfection of an OBG3 fragment-expressing vector into mouse NIH 3T3cells is one embodiment of introducing polynucleotides into host cells.Introduction of a polyiiucleotide encoding a polypeptide into a hostcell can be effected by calcium phosphate transfection, DEAE-dextranmediated transfection, cationic lipid-mediated transfection,electroporation, transduction, infection, or other methods. Such methodsare described in many standard laboratory manuals, such as Davis et al.((1986) Methods in Molecular Biology, Elsevier Science Publishing Co.,Inc., Amsterdam). It is specifically contemplated that the polypeptidesof the present invention may in fact be expressed by a host cell lackinga recombinant vector. Methods of expressing OBG3 fragment of theinvention in cells are described in Examples 1-3.

A polypeptide of this invention (i.e. a gOBG3 fragment) can be recoveredand purified from recombinant cell cultures by well-known methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention, and preferably the secreted form, can also be recovered from:products purified from natural sources, including bodily fluids, tissuesand cells, whether directly isolated or cultured; products of chemicalsynthetic procedures; and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect, and mammalian cells.

Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. Preferably OBG3 globular domain comprising thepolypeptides of the invention is non-glycosylated. In addition,polypeptides of the invention may also include an initial modifiedmethionine residue, in some cases as a result of host-mediatedprocesses. Thus, it is well known in the art that the N-terminalmethionine encoded by the translation initiation codon generally isremoved with high efficiency from any protein after translation in alleukaryotic cells. While the N-terminal methionine on most proteins alsois efficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination, see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,(1989) Proc Natl Acad Sci USA 86(22):8932-5; Koller et al., (1989) ProcNatl Acad Sci USA 86(22):8927-31; and Zijlstra et al. (1989) Nature342(6248):435-8; the disclosures of each of which are incorporated byreference in their entireties).

Modifications

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (See, e.g., Creighton, 1983 Proteins.New York, N.Y.: W.H. Freeman and Company; and Hunkapiller et al., (1984)Nature 310(5973):105-1 1). For example, a relative short fragment of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thefragment sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptide fragments which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptide fragments may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the polypeptide.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention that may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity. See U.S. Pat. No: 4,179,337.The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the polypeptide with consideration of effects on functionalor antigenic domains of the polypeptide. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al. (1992) Exp Hematol 20(8):1028-35, reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus may be accomplished by reductive alkylation,which exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

Multimers

The polypeptide fragments of the invention may be in monomers ormultimers. Most preferably, the polypeptide fragments of the inventionare in homotrimers. Accordingly, the present invention relates tomonomers and multimers of the polypeptide fragments of the invention,their preparation, and compositions (preferably, pharmaceutical orphysiologically acceptable compositions) containing them. In specificembodiments, the polypeptides of the invention are homotrimers. Inadditional embodiments, the multimers of the invention comprise, consistessentially of, or consist of homotrimers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer refers to a multimer containing onlypolypeptides corresponding to the OBG3 polypeptide fragments of theinvention (including polypeptide fragments, variants, splice variants,and fusion proteins corresponding to these polypeptide fragments asdescribed herein). These homomers may contain polypeptide fragmentshaving identical or different amino acid sequences. In a specificembodiment, a homomer of the invention is a multimer containing onlypolypeptide fragments having an identical amino acid sequence. Inanother specific embodiment, a homomer of the invention is a multimercontaining polypeptide fragments having different amino acid sequences.In specific embodiments, the multimer of the invention is a homodimer(e.g., containing polypeptide fragments having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptidefragments having identical and/or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.More preferably, the homomeric multimer of the invention is ahomotrimer. Further more preferably, said homotrimer of the invention isgOBG3 polypeptide fragment homotrimer. Most preferably, said gOBG3polypeptide fragment homotrimer of the invention has activity selectedfrom the group consisting of lipid partitioning, lipid metabolism; andinsulin-like activity. Also most preferably, said gOBG3 polypeptidefragment homotrimer of the invention has activity selected from thegroup consisting of prevention of weight gain, weight reduction, andmaintenance of weight loss.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., corresponding to differentproteins or polypeptide fragments thereof) in addition to thepolypeptides of the invention. In a specific embodiment, the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the heteromeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in most preferredembodiment, homotrimers of the invention are formed when polypeptides ofthe invention contact one another in solution. In another embodiment,heteromultimers of the invention, such as, for example, heterotrimers orheterotetramers, are formed when polypeptides of the invention contactantibodies to the polypeptides of the invention (including antibodies tothe heterologous polypeptide sequence in a fusion protein of theinvention) in solution. In other embodiments, multimers of the inventionare formed by covalent associations with and/or between the polypeptidesof the invention. Such covalent associations may involve one or moreamino acid residues contained in the polypeptide sequence (e.g., thatrecited in the sequence listing, or contained in the polypeptide encodedby a deposited clone). In one instance, the covalent associations arecross-linking between cysteine residues located within the polypeptidesequences, which interact in the native (i.e., naturally occurring)polypeptide. In another instance, the covalent associations are theconsequence of chemical or recombinant manipulation. Alternatively, suchcovalent associations may involve one or more amino acid residuescontained in the heterologous polypeptide sequence in a fusion proteinof the invention.

In particularly preferred embodiment of the invention, said multimers ofthe invention are gOBG3 polypeptide fragment homotrimers. Mostpreferably, said gOBG3 polypeptide fragment homotrimer of the inventionhas activity selected from the group consisting of lipid partitioning,lipid metabolism, and insulin-like activity. Also most preferably, saidgOBG3 polypeptide fragment homotrimer of the invention has activityselected from the group consisting of prevention of weight gain, weightreduction, and maintenance of weight loss.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins, and have since been found ina variety of different proteins (Landschulz et al., (1988) Genes Dev.July; 2(7):786-800). Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solublemultimeric proteins of the invention are those described in PCTapplication WO 94/10308, hereby incorporated by reference. Recombinantfusion proteins comprising a polypeptide of the invention fused to apolypeptide sequence that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble multimericfusion protein is recovered from the culture supernatant usingtechniques known in the art

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. FEBS Letters (1994) 344(2-3):191-5and in U.S. patent application Ser. No.08/446,922, hereby incorporatedby reference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention. In another example, proteins of the invention are associatedby interactions between Flag® & polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, proteins of the invention are associated byinteractions between heterologous polypeptide sequence contained inFlag® fusion proteins of the invention and anti Flag® antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C-terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, at least 30techniques known in the art may be applied to generate liposomescontaining the polypeptide components desired to be contained in themultimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (See, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

II. OBG3 Polynucleotides of the Invention

Preferred polynucleotides are those that encode full-length OBG3 andgOBG3 polypeptide fragments of the invention. The recombinantpolynucleotides encoding full-length OBG3 and gOBG3 polypeptidefragments can be used in a variety of ways, including, but not limitedto, expressing the polypeptide in recombinant cells for use in screeningassays for antagonists and agonists of its activity as well as tofacilitate its purification for use in a variety of ways including, butnot limited to screening assays for agonists and antagonists of itsactivity, diagnostic screens, and raising antibodies, as well astreatment and/or prevention of obesity-related diseases and disordersand/or to reduce body mass.

The invention relates to the polynucleotides encoding full-length OBG3and gOBG3 polypeptide fragments and variant polypeptide fragmentsthereof as described herein. These polynucleotides may be purified,isolated, and/or recombinant. In all cases, the desired OBG3 and gOBG3polynucleotides of the invention are those that encode gOBG3 polypeptidefragments of the invention having obesity-related activity as describedand discussed herein.

Fragments

A polynucleotide fragment is a polynucleotide having a sequence thatentirely is the same as part, but not all, of the full-length OBG3polypeptide or a specified gOBG3 polypeptide nucleotide sequence. Suchfragments may be “free-standing”, i.e. not part of or fused to otherpolynucleotides, or they may be comprised within another non-OBG3 ornon-gOBG3 (heterologous) polynucleotide of which they form a part orregion. However, several gOBG3 polynucleotide fragments may be comprisedwithin a single polynucleotide.

The OBG3 polynucleotides of the invention comprise from 18 consecutivebases to 18 consecutive bases less than the full-length polynucleotidesequence encoding the intact OBG3 polypeptide, for example thefull-length OBG3 polypeptide polynucleotide sequences in SEQ ID NO: 1 orSEQ ID NO:3. In one aspect of this embodiment, the polynucleotidecomprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570,575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710,715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780,785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920,925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990,995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050,1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110,1115, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170,1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230,1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290,1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350,1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410,1415, 1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470,1475, 1480, 1485, 1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530,1535, 1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590,1595, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635, 1640, 1645, 1650,1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710,1715, 1720, 1725, 1730, 1735, 1740, 1745, 1750, 1755, 1760, 1765, 1770,1775, 1780, 1785, 1790, 1795, 1800, 1805, 1810, 1815, 1820, 1825, 1830,1835, 1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890,1895, 1900, 1905, 1910, 1915, 1920, 1925, 1930, 1935, 1940, 1945, 1950,1955, 1960, 1965, 1970, 1975, 1980, 1985, 1990, 1995, 2000, 2005, 2010,2015, 2020, 2025, 2030, 2035, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, 2095, 2100, 2105, 2110, 2115, 2120, 2125, 2130,2135, 2140, 2145, 2150, 2155, 2160, 2165, 2170, 2175, 2180, 2185, 2190,2195, 2200, 2205, 2210, 2215, 2220, 2225, 2230, 2235, 2240, 2245, 2250,2255, 2260, 2265, 2270, 2275, 2280, 2285, 2290, 2295, 2300, 2305, 2310,2315, 2320, 2325, 2330, 2335, 2340, 2345, 2350, 2355, 2360, 2365, 2370,2375, 2380, 2385, 2390, 2395, 2400, 2405, 2410, 2415, 2420, 2425, 2430,2435, 2440, 2445, 2450, 2455, 2460, 2465, 2470, 2475, 2480, 2485, 2490,2495, 2500, 2505, 2510, 2515, 2520, 2525, 2530, 2535, 2540, 2545, 2550,2555, 2560, 2565, 2570, 2575, 2580, 2585, 2590, 2595, 2600, 2605, 2610,2615, 2620, 2625, 2630, 2635, 2640, 2645, 2650, 2655, 2660, 2665, 2670,2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720, 2725, 2730,2735, 2740, 2745, 2750, 2755, 2760, 2765, 2770, 2775, 2780, 2785, 2790,2795, 2800, 2805, 2810, 2815, 2820, 2825, 2830, 2835, 2840, 2845, 2850,2855, 2860, 2865, 2870, 2875, 2880, 2885, 2890, 2895, 2900, 2905, 2910,2915, 2920, 2925, 2930, 2935, 2940, 2945, 2950, 2955, 2960, 2965, 2970,2975, 2980, 2985, 2990, 2995, 3000, 3005, 3010, 3015, 3020, 3025, 3030,3035, 3040, 3045, 3050, 3055, 3060, 3065, 3070, 3075, 3080, 3085, 3090,3095, 3100, 3105, 3110, 3115, 3120, 3125, 3130, 3135, 3140, 3145, 3150,3155, 3160, 3165, 3170, 3175, 3180, 3185, 3190, 3195, 3200, 3205, 3210,3215, 3220, 3225, 3230, 3235, 3240, 3245, 3250, 3255, 3260, 3265, 3270,3275, 3280, 3285, 3290, 3295, 3300, 3305, 3310, 3315, 3320, 3325, 3330,3335, 3340, 3345, 3350, 3355, 3360, 3365, 3370, 3375, 3380, 3385, 3390,3395, 3400, 3405, 3410, 3415, 3420, 3425, 3430, 3435, 3440, 3445, 3450,3455, 3460, 3465, 3470, 3475, 3480, 3485, 3490, 3495, 3500, 3505, 3510,3515, 3520, 3525, 3530, 3535, 3540, 3545, 3550, 3555, 3560, 3565, 3570,3575, 3580, 3585, 3590, 3595, 3600, 3605, 3610, 3615, 3620, 3625, 3630,3635, 3640, 3645, 3650, 3655, 3660, 3665, 3670, 3675, 3680, 3685, 3690,3695, 3700, 3705, 3710, 3715, 3720, 3725, 3730, 3735, 3740, 3745, 3750,3755, 3760, 3765, 3770, 3775, 3780, 3785, 3790, 3795, 3800, 3805, 3810,3815, 3820, 3825, 3830, 3835, 3840, 3845, 3850, 3855, 3860, 3865, 3870,3875, 3880, 3885, 3890, 3895, 3900, 3905, 3910, 3915, 3920, 3925, 3930,3935, 3940, 3945, 3950, 3955, 3960, 3965, 3970, 3975, 3980, 3985, 3990,3995, 4000, 4005, 4010, 4015, 4020, 4025, 4030, 4035, 4040, 4045, 4050,4055, 4060, 4065, 4070, 4075, 4080, 4085, 4090, 4095, 4100, 4105, 4110,4115, 4120, 4125, 4130, 4135, 4140, 4145, 4150, 4155, 4160, 4165, 4170,4175, 4180, 4185, 4190, 4195, 4200, 4205, 4210, 4215, 4220, 4225, 4230,4235, 4240, 4245, 4250, 4255, 4260, 4265, 4270, 4275, 4280, 4285, 4290,4295, 4300, 4305, 4310, 4315, 4320, 4325, 4330, 4335, 4340, 4345, 4350,4355, 4360, 4365, 4370, 4375, 4380, 4385, 4390, 4395, 4400, 4405, 4410,4415, 4420, 4425, 4430, 4435, 4440, 4445, 4450, 4455, 4460, 4465, 4470,4475, 4480, 4485, 4490, 4495, 4500, 4505, 4510 or 4515 consecutivenucleotides of a polynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18 nucleotides, wherein “at least 18” isdefined as any integer between 18 and the integer representing the 3′most nucleotide position of the intact OBG3 polypeptide cDNA as setforth in the sequence listing (SEQ D NO:1 or SEQ D NO:3) or elsewhereherein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the ORF, i.e., the nucleotide “A” of the start codon (ATG)with the remaining nucleotides numbered consecutively. Therefore, everycombination of a 5′ and 3′ nucleotide position that a polynucleotidefragment invention, at least 18 contiguous nucleotides in length, couldoccupy on an intact OBG3 polypeptide polynucleotide of the presentinvention is included in the invention as an individual species. Thepolynucleotide fragments specified by 5′ and 3′ positions can beimmediately envisaged and are therefore not individually listed solelyfor the purpose of not unnecessarily lengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of thepolynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 19 and the number of nucleotides of thepolynucleotide sequence of the present invention; and where “x” is aninteger smaller then “y” by at least 18.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above.

The gOBG3 polynucleotide fragments of the invention comprise from 18consecutive bases to the full-length polynucleotide sequence encodingthe gOBG3 fragments described in Section II of the Preferred Embodimentsof the Invention. In one aspect of this embodiment, the polynucleotidecomprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570,575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640,645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710,715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780,785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920,925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990,995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050,1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110,1115, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170,1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230,1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290,1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350,1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410,1415, 1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470,1475, 1480, 1485, 1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530,1535, 1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590,1595, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635, 1640, 1645, 1650,1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1705, 1710,1715, 1720, 1725, 1730, 1735, 1740, 1745, 1750, 1755, 1760, 1765, 1770,1775, 1780, 1785, 1790, 1795, 1800, 1805, 1810, 1815, 1820, 1825, 1830,1835, 1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890,1895, 1900, 1905, 1910, 1915, 1920, 1925, 1930, 1935, 1940, 1945, 1950,1955, 1960, 1965, 1970, 1975, 1980, 1985, 1990, 1995, 2000, 2005, 2010,2015, 2020, 2025, 2030, 2035, 2040, 2045, 2050, 2055, 2060, 2065, 2070,2075, 2080, 2085, 2090, 2095, 2100, 2105, 2110, 2115, 2120, 2125, 2130,2135, 2140, 2145, 2150, 2155, 2160, 2165, 2170, 2175, 2180, 2185, 2190,2195, 2200, 2205, 2210, 2215, 2220, 2225, 2230, 2235, 2240, 2245, 2250,2255, 2260, 2265, 2270, 2275, 2280, 2285, 2290, 2295, 2300, 2305, 2310,2315, 2320, 2325, 2330, 2335, 2340, 2345, 2350, 2355, 2360, 2365, 2370,2375, 2380, 2385, 2390, 2395, 2400, 2405, 2410, 2415, 2420, 2425, 2430,2435, 2440, 2445, 2450, 2455, 2460, 2465, 2470, 2475, 2480, 2485, 2490,2495, 2500, 2505, 2510, 2515, 2520, 2525, 2530, 2535, 2540, 2545, 2550,2555, 2560, 2565, 2570, 2575, 2580, 2585, 2590, 2595, 2600, 2605, 2610,2615, 2620, 2625, 2630, 2635, 2640, 2645, 2650, 2655, 2660, 2665, 2670,2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720, 2725, 2730,2735, 2740, 2745, 2750, 2755, 2760, 2765, 2770, 2775, 2780, 2785, 2790,2795, 2800, 2805, 2810, 2815, 2820, 2825, 2830, 2835, 2840, 2845, 2850,2855, 2860, 2865, 2870, 2875, 2880, 2885, 2890, 2895, 2900, 2905, 2910,2915, 2920, 2925, 2930, 2935, 2940, 2945, 2950, 2955, 2960, 2965, 2970,2975, 2980, 2985, 2990, 2995, 3000, 3005, 3010, 3015, 3020, 3025, 3030,3035, 3040, 3045, 3050, 3055, 3060, 3065, 3070, 3075, 3080, 3085, 3090,3095, 3100, 3105, 3110, 3115, 3120, 3125, 3130, 3135, 3140, 3145, 3150,3155, 3160, 3165, 3170, 3175, 3180, 3185, 3190, 3195, 3200, 3205, 3210,3215, 3220, 3225, 3230, 3235, 3240, 3245, 3250, 3255, 3260, 3265, 3270,3275, 3280, 3285, 3290, 3295, 3300, 3305, 3310, 3315, 3320, 3325, 3330,3335, 3340, 3345, 3350, 3355, 3360, 3365, 3370, 3375, 3380, 3385, 3390,3395, 3400, 3405, 3410, 3415, 3420, 3425, 3430, 3435, 3440, 3445, 3450,3455, 3460, 3465, 3470, 3475, 3480, 3485, 3490, 3495, 3500, 3505, 3510,3515, 3520, 3525, 3530, 3535, 3540, 3545, 3550, 3555, 3560, 3565, 3570,3575, 3580, 3585, 3590, 3595, 3600, 3605, 3610, 3615, 3620, 3625, 3630,3635, 3640, 3645, 3650, 3655, 3660, 3665, 3670, 3675, 3680, 3685, 3690,3695, 3700, 3705, 3710, 3715, 3720, 3725, 3730, 3735, 3740, 3745, 3750,3755, 3760, 3765, 3770, 3775, 3780, 3785, 3790, 3795, 3800, 3805, 3810,3815, 3820, 3825, 3830, 3835, 3840, 3845, 3850, 3855, 3860, 3865, 3870,3875, 3880, 3885, 3890, 3895, 3900, 3905, 3910, 3915, 3920, 3925, 3930,3935, 3940, 3945, 3950, 3955, 3960, 3965, 3970, 3975, 3980, 3985, 3990,3995, 4000, 4005, 4010, 4015, 4020, 4025, 4030, 4035, 4040, 4045, 4050,4055, 4060, 4065, 4070, 4075, 4080, 4085, 4090, 4095, 4100, 4105, 4110,4115, 4120, 4125, 4130, 4135, 4140, 4145, 4150, 4155, 4160, 4165, 4170,4175, 4180, 4185, 4190, 4195, 4200, 4205, 4210, 4215, 4220, 4225, 4230,4235, 4240, 4245, 4250, 4255, 4260, 4265, 4270, 4275, 4280, 4285, 4290,4295, 4300, 4305, 4310, 4315, 4320, 4325, 4330, 4335, 4340, 4345, 4350,4355, 4360, 4365, 4370, 4375, 4380, 4385, 4390, 4395, 4400, 4405, 4410,4415, 4420, 4425, 4430, 4435, 4440, 4445, 4450, 4455, 4460, 4465, 4470,4475, 4480, 4485, 4490, 4495, 4500, 4505, 4510 or4515 consecutivenucleotides of a polynucleotide of the present invention.

In addition to the above preferred nucleic acid sizes, further preferrednucleic acids comprise at least 18. nucleotides, wherein “at least 18”is defined as any integer between 18 and the integer corresponding tothe 3′ most nucleotide position of a gOBG3 fragment cDNA herein.

Further included as preferred polynucleotides of the present inventionare nucleic acid fragments at least 18 nucleotides in length, asdescribed above, that are further specified in terms of their 5′ and 3′position. The 5′ and 3′ positions are represented by the positionnumbers set forth in the sequence listing below. For allelic anddegenerate and other variants, position 1 is defined as the 5′ mostnucleotide of the open reading frame (ORF), i.e., the nucleotide “A” ofthe start codon (ATG) with the remaining nucleotides numberedconsecutively. Therefore, every combination of a 5′ and 3′ nucleotideposition that a polynucleotide fragment invention, at least 18contiguous nucleotides in length, could occupy on a gOBG3 fragmentpolynucleotide of the present invention is included in the invention asan individual species. The polynucleotide fragments specified by 5′ and3′ positions can be immediately envisaged and are therefore notindividually listed solely for the purpose of not unnecessarilylengthening the specification.

It is noted that the above species of polynucleotide fragments of thepresent invention may alternatively be described by the formula “x toy”; where “x” equals the 5′ most nucleotide position and “y” equals the3′ most nucleotide position of the polynucleotide; and further where “x”equals an integer between 1 and the number of nucleotides of the gOBG3polynucleotide sequence of the present invention minus 18, and where “y”equals an integer between 9 and the number of nucleotides of the gOBG3polynucleotide sequence of the present invention; and where “x” is aninteger smaller than “y” by at least 18. Every combination of “x” and“y” positions are included as specific embodiments of the invention.Moreover, the formula “x” to “y” may be modified as ‘“x1-x2” to“y1-y2”’, wherein “x1-x2” and “y1-y2” represent positional rangesselected from any two nucleotide positions of the sequence listing.Alterative formulas include ‘“x1-x2” to “y”’ and ‘“x” to “y1-y2”’.

These specific embodiments, and other polynucleotide fragmentembodiments described herein may be modified as being “at least”, “equalto”, “equal to or less than”, “less than”, “at least ______ but notgreater than ______” or “from ______ to ______” a specified size orspecified 5′ and/or 3′ positions.

The present invention also provides for the exclusion of any species ofpolynucleotide fragments of the present invention specified by 5′ and 3′positions or polynucleotides specified by size in nucleotides asdescribed above.

Variants

In other preferred embodiments, variants of gOBG3 polynucleotidesencoding gOBG3 polypeptide fragments are envisioned. Variants ofpolynucleotides, as the term is used herein, are polynucleotides whosesequence differs from a reference polynucleotide. A variant of apolynucleotide may be a naturally occurring variant such as a naturallyoccurring allelic variant, or it may be a variant that is not known tooccur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.

Polynucleotide variants that comprise a sequence substantially differentfrom those described above but that, due to the degeneracy of thegenetic code, still encode gOBG3 polypeptide fragments of the presentinvention are also specifically envisioned. It would also be routine forone skilled in the art to generate the degenerate variants describedabove, for instance, to optimize codon expression for a particular host(e.g. change codons in the human mRNA to those preferred by othermammalian or bacterial host cells).

As stated above, variant polynucleotides may occur naturally, such as anatural allelic variant, or by recombinant methods. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism (See, e.g., B. Lewin,(1990) Genes IV, Oxford University Press, New York). Non-naturallyoccurring variants may be produced using art-known mutagenesistechniques. Such nucleic acid variants include those produced bynucleotide substitutions, deletions, or additions. The substitutions,deletions, or additions may involve one or more nucleotides. Alterationsin the coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of a gOBG3 polypeptide fragment ofthe invention. Also preferred in this regard are conservativesubstitutions.

Nucleotide changes present in a variant polynucleotide are preferablysilent, which means that they do not alter the amino acids encoded bythe polynucleotide. However, nucleotide changes may also result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence.

In cases where the nucleotide substitutions result in one or more aminoacid changes, preferred gOBG3 polypeptide fragments include those thatretain one or more obesity-related activity as described in Section I ofthe Preferred Embodiments of the Invention.

By “retain the same activities” is meant that the activity measuredusing the polypeptide encoded by the variant gOBG3 polynucleotide inassays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%,and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120% or 125%of the activity measured using a gOBG3 fragment described in theExamples Section herein.

By the activity being “increased” is meant that the activity measuredusing the polypeptide encoded by the variant gOBG3 polynucleotide inassays is at least 125%, 130%, 135%, 140%, 145%, 150%,155%, 160%, 170%,180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%,or 500% of the activity measured using a gOBG3 fragment described in theExamples Section herein.

By the activity being “decreased” is meant that the activity measuredusing the polypeptide encoded by the variant gOBG3 polynucleotide inassays is decreased by at least 25%, 30%, 35%, 40%, 45%, or 50% of theactivity measured using a gOBG3 fragment described in the ExamplesSection herein.

Percent Identity

The present invention is further directed to nucleic acid moleculeshaving sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or99% identical to the polynucleotide sequences of SEQ ID NO:1 or SEQ IDNO:3 or fragments thereof that encode a polypeptide havingobesity-related activity as described in Section I of the PreferredEmbodiments of the Invention. Of course, due to the degeneracy of thegenetic code, one of ordinary skill in the art will immediatelyrecognize that a large number of the nucleic acid molecules at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequences shown in SEQ ID NO:1 or SEQ ID NO:3 or fragmentsthereof will encode a polypeptide having biological activity. In fact,since degenerate variants of these nucleotide sequences all encode thesame polypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving biological activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly affect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed previously in Section I of the Preferred Embodiments of theInvention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the gOBG3fragment. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted,inserted, or substituted with another nucleotide. The query sequence maybe an entire sequence or any fragment specified as described herein.

The methods of determining and defining whether any particular nucleicacid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to a nucleotide sequence of the presentinvention can be done by using known coniputer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al.,((1990) Comput Appl Biosci 6(3):23745). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by first converting U's to T's. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matchedlaligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90-nucleotide subject sequence is aligned to a100-nucleotide query sequence to determine percent identity. Thedeletions occur at the 5′ end of the subject sequence and therefore, theFASTDB alignment does not show a matched/alignment of the first 10nucleotides at 5′ end. The 10 unpaired nucleotides represent 10% of thesequence (number of nucleotides at the 5′ and 3′ ends not matched/totalnumber of nucleotides in the query sequence) so 10% is subtracted fromthe percent identity score calculated by the FASTDB program. If theremaining 90 nucleotides were perfectly matched the final percentidentity would be 90%.

In another example, a 90 nucleotide subject sequence is compared with a100 nucleotide query sequence. This time the deletions are internaldeletions so that there are no nucleotides on the 5′ or 3′ of thesubject sequence which are not matched/aligned with the query. In thiscase the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made for thepurposes of the present invention.

Fusions

Further included in the present invention are polynucleotides encodingthe polypeptides of the present invention that are fused in frame to thecoding sequences for additional heterologous amino acid sequences. Alsoincluded in the present invention are nucleic acids encodingpolypeptides of the present invention together with additional,non-coding sequences, including for example, but not limited tonon-coding 5′ and 3′ sequences, vector sequence, sequences used forpurification, probing, or priming. For example, heterologous sequencesinclude transcribed, nontranslated sequences that may play a role intranscription, and mRNA processing, for example, ribosome binding andstability of mRNA. The heterologous sequences may alternatively compriseadditional coding sequences that provide additional functionalities.Thus, a nucleotide sequence encoding a polypeptide may be fused to a tagsequence, such as a sequence encoding a peptide that facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the tag amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. For instance,hexa-histidine provides for convenient purification of the fusionprotein (See, Gentz et al., (1989) Proc Natl Acad Sci USA 86(3):821-4).The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein (See, Wilson et al., (1984) Cell 37(3):767-78). As discussedabove, other such fusion proteins include gOBG3 fragment cDNA fused toFc at the N— or C-terminus.

III. Recombinant Vectors of the Invention

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, that is either double-stranded orsingle-stranded, and that comprises at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

The present invention relates to recombinant vectors comprising any oneof the polynucleotides described herein.

The present invention encompasses a family of recombinant vectors thatcomprise polynucleotides encoding OBG3 polypeptide fragments of theinvention.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide in a suitable cell host,this polynucleotide being amplified every time that the recombinantvector replicates. The inserted polynucleotide can be one that encodesgOBG3 polypeptide fragments of the invention.

A second preferred embodiment of the recombinant vectors according tothe invention consists of expression vectors comprising polynucleotidesencoding OBG3 polypeptide fragments of the invention. Within certainembodiments, expression vectors are employed to express an OBG3 fragmentof the invention, preferably a modified OBG3 fragment described in thepresent invention, which can be then purified and, for example, be usedas a treatment for obesity-related diseases, or simply to reduce bodymass of individuals.

Expression requires that appropriate signals are provided in thevectors, said signals including various regulatory elements, such asenhancers/promoters from both viral and mammalian sources, that driveexpression of the genes of interest in host cells. Dominant drugselection markers for establishing permanent, stable, cell clonesexpressing the products are generally included in the expression vectorsof the invention, as they are elements that link expression of the drugselection markers to expression of the polypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding an OBG3 fragment of the invention,or a modified OBG3 fragment as described herein, or variants orfragments thereof, under the control of a regulatory sequence selectedamong OBG3 polypeptide fragments, or alternatively under the control ofan exogenous regulatory sequence.

Consequently, preferred expression vectors of the invention are selectedfrom the group consisting of: (a) an OBG3 fragment regulatory sequenceand driving the expression of a coding polynucleotide operably linkedthereto; and (b) an OBG3 fragment coding sequence of the invention,operably linked to regulatory sequences allowing its expression in asuitable cell host and/or host organism.

Some of the elements that can be found in the vectors of the presentinvention are described in further detail in the following sections.

1) General Features of the Expression Vectors of the Invention:

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, oreven a linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic or synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in geneexpression, for example promoters or enhancers. Enhancers are cis-actingelements of DNA, usually from about 10 to 300 bp in length that act onthe promoter to increase the transcription;

(2) a structural or coding sequence which is transcribed into mRNA andeventually translated into a polypeptide, said structural or codingsequence being operably linked to the regulatory elements described in(1); and

(3) appropriate transcription initiation and termination sequences.Structural units intended for use in yeast or eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, when arecombinant protein is expressed without a leader or transport sequence,it may include a N-terminal residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsites, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

2) Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors of thepresent invention are chosen taking into account the cell host in whichthe heterologous gene is expressed. The particular promoter employed tocontrol the expression of a nucleic acid sequence of interest is notbelieved to be important, so long as it is capable of directing theexpression of the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell, such as, for example, a human or aviral promoter. The promoter used may be constitutive or inducible.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors. Preferred bacterial promoters are the LacI,LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt,lambda PR, PL and trp promoters (EP 0036776), the polyhedrin promoter,or the p10 protein promoter from baculovirus (Kit Novagen) (Smith etal., (1983) Mol Cell Biol 3(12):2156-65; O'Reilly et al., 1992), thelambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus., and mouse metallothionein-L.In addition, promoters specific for a particular cell type may bechosen, such as those facilitating expression in adipose tissue, muscletissue, or liver. Selection of a convenient vector and promoter is wellwithin the level of ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic engineering. For example, one may refer toSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), or also to theprocedures described by Fuller et al. (1996) immunology in CurrentProtocols in Molecular Biology.

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

Vectors containing the appropriate DNA sequence as described above canbe utilized to transform an appropriate host to allow the expression ofthe desired polypeptide or polynucleotide.

3) Selectable Markers

Such markers would confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

4) Preferred Vectors

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, but are not limited to, pKK223-3 (Pharmacia, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Large numbers of other suitable vectors are known to those of skill inthe art, and are commercially available, such as the following bacterialvectors: pTrc-His, pET30-His, pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10,phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3,pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).

Baculovirus Vectors

A suitable vector for the expression of polypeptides of the invention isa baculovirus vector that can be propagated in insect cells and ininsect cell lines. A specific suitable host vector system is thepVL1392/1393 baculovirus transfer vector (Pharmingen) that is used totransfect the SF9 cell line (ATCC N° CRL 1711) which is derived fromSpodoptera frugiperda.

Further suitable baculovirus vectors are known to those skilled in theart, for example, FastBacHT. Other suitable vectors for the expressionof an APM1 globular head polypeptide in a baculovirus expression systeminclude, but are not limited to, those described by Chai et al. (1993;Biotechnol Appl Biochem. December 18 (Pt 3):259-73); Vlasak et al.(1983; Eur J Biochem September 1;135(1):123-6); and Lenhard et al.(1996; Gene March 9;169(2):187-90).

Mammalian Vectors

Further suitable vectors for the expression of polypeptides of theinvention are mammalian vectors. A number of suitable vector systems areknown to those skilled in the art, for example, pcDNA4HisMax,pcDNA3.1Hygro-His and pcDNA3.1Hygro.

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996; Semin Interv Cardiol 1(3):203-8) orOhno et al. (1994; Science 265(5173):7814). Another preferredrecombinant adenovirus according to this specific embodiment of thepresent invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) oran adenovirus of animal origin (French patent application No.FR-93.05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vivo gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., ((1989) Proc Natl Acad Sci USA86(23):9079-83), Julan et al., (1992) J. Gen. Virol. 3:3251-3255 andNeda et al. ((1991) J Biol Chem 266(22):14143-6). Yet another viralvector system that is contemplated by the invention consists of theadeno-associated virus (AAV). The adeno-associated virus is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., (1992) CurrTop Microbiol Immunol 158:97-129). It is also one of the few virusesthat may integrate its DNA into non-dividing cells, and exhibits a highfrequency of stable integration (Flotte et al., (1992) Am J Respir CellMol Biol 7(3):349-56; Samulski et al., (1989) J Virol 63(9):3822-8;McLaughlin et al., (1989) An J Hum Genet 59:561-569). One advantageousfeature of AAV derives from its reduced efficacy for transducing primarycells relative to transformed cells.

5) Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides of the invention,these constructs must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transforming celllines, or in vivo or ex vivo, as in the treatment of certain diseasestates.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calcium phosphate precipitation(Graham et al., (1973) Virology 54(2):536-9; Chen et al., (1987) MolCell Biol 7(8):2745-52), DEAE-dextran (Gopal, (1985) Mol Cell Biol5(5):1188-90), electroporation (Tur-Kaspa et al., (1986) Mol Cell Biol6(2):716-8; Potter et al., (1984) Proc Natl Acad Sci USA81(22):7161-5.), direct microinjection (Harland et al., (1985) J CellBiol 101(3):1094-9), DNA-loaded liposomes (Nicolau et al., (1982)Biochim. Biophys Acta 721(2):185-90; Fraley et al., (1979) Proc NatlAcad Sci USA 76(7):3348-52), and receptor-mediated transfection (Wu andWu, (1987) J Biol Chem 262(10):4429-32; Wu and Wu (1988) Biochemistry27(3):887-92). Some of these techniques may be successfully adapted forin vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No. WO 95/11307 (Institut Pasteur,INSERM, Université d'Ottawa) as well as in the articles of Tascon et al.(1996) Nature Medicine 2(8):888-892 and of Huygen et al. ((1996) Nat Med2(8):893-8).

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.((1990) Curr Genet Feb;17(2):97-103).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther4:87-103; Wong et al., (1980) Gene 10:87-94; Nicolau et al., (1987)Methods Enzymol 149:157-76). These liposomes may further be targeted tocells expressing LSR by incorporating leptin, triglycerides, ACRP30, orother known LSR ligands into the liposome membrane.

In a specific embodiment, the invention provides a composition for thein vivo production of an APM1 globular head polypeptide describedherein. It comprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said polypeptide.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body. In anotherembodiment of the vector according to the invention, it may beintroduced in vitro in a host cell, preferably in a host cell previouslyharvested from the animal to be treated and more preferably a somaticcell such as a muscle cell. In a subsequent step, the cell that has beentransformed with the vector coding for the desired APM1 globular headpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

IV. Recombinant Cells of the Invention

Another object of the invention consists of host cells recombinant for,i.e., that have been transformed or transfected with one of thepolynucleotides described herein, and more precisely a polynucleotidecomprising a polynucleotide encoding an OBG3 polypeptide fragment of theinvention such as any one of those described in “Polynucleotides of theInvention”. These polynucleotides can be present in cells as a result oftransient or stable transfection. The invention includes host cells thatare transformed (prokaryotic cells) or that are transfected (eukaryoticcells) with a recombinant vector such as any one of those described in“Recombinant Vectors of the Invention”.

Generally, a recombinant host cell of the invention comprises at leastone of the polynucleotides or the recombinant vectors of the inventionthat are described herein.

Preferred host cells used as recipients for the recombinant vectors ofthe invention are the following:

a) Prokaryotic host cells: Escherichia coli strains (I.E. DH5-x strain),Bacillus subtilis, Salmonella typhimurium, and strains from species likePseudomonas, Streptomyces and Staphylococcus, and

b) Eukaryotic host cells: HeLa cells (ATCC N° CCL2; N° CCL2.1; N°CCL2.2), Cv 1 cells (ATCC N° CCL70), COS cells (ATCC N° CRL1650; N°CRL1651), Sf-9 cells (ATCC N° CRL1711), C127 cells (ATCC N° CRL-1804),3T3 (ATCC N° CRL-6361), CHO (ATCC N° CCL-61), human kidney 293 (ATCC N°45504; N° CRL-1573), BHK (ECACC N° 84100501; N° 84111301), PLC cells,HepG2, and Hep3B.

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

Further, according to the invention, these recombinant cells can becreated in vitro or in vivo in an animal, preferably a manual, mostpreferably selected from the group consisting of mice, rats, dogs, pigs,sheep, cattle, and primates, not to include humans. Recombinant cellscreated in vitro can also be later surgically implanted in an animal,for example. Methods to create recombinant cells in vivo in animals arewell known in the art.

The present invention also encompasses primary, secondary, andimmortalized homologously recombinant host cells of vertebrate origin,preferably mammalian origin and particularly human origin, that havebeen engineered to: a) insert exogenous (heterologous) polynucleotidesinto the endogenous chromosomal DNA of a targeted gene, b) deleteendogenous chromosomal DNA, and/or c) replace endogenous chromosomal DNAwith exogenous polynucleotides. Insertions, deletions, and/orreplacements of polynucleotide sequences may be to the coding sequencesof the targeted gene and/or to regulatory regions, such as promoter andenhancer sequences, operably associated with the targeted gene.

The present invention further relates to a method of making ahomologously recombinant host cell in vitro or in vivo, wherein theexpression of a targeted gene not normally expressed in the cell isaltered. Preferably the alteration causes expression of the targetedgene under normal growth conditions or under conditions suitable forproducing the polypeptide encoded by the targeted gene. The methodcomprises the steps of: (a) transfecting the cell in vitro or in vivowith a polynucleotide construct, the polynucleotide constructcomprising; (i) a targeting sequence; (ii) a regulatory sequence and/ora coding sequence; and (iii) an unpaired splice donor site, ifnecessary, thereby producing a transfected cell; and (b) maintaining thetransfected cell in vitro or in vivo under conditions appropriate forhomologous recombination.

The present invention further relates to a method of altering theexpression of a targeted gene in a cell in vitro or in vivo wherein thegene is not normally expressed in the cell, comprising the steps of: (a)transfecting the cell in vitro or in vivo with a polynucleotideconstruct, the polynucleotide construct comprising: (i) a targetingsequence; (ii) a regulatory sequence and/or a coding sequence; and (iii)an unpaired splice donor site, if necessary, thereby producing atransfected cell; and (b) maintaining the transfected cell in vitro orin vivo under conditions appropriate for homologous recombination,thereby producing a homologously recombinant cell; and (c) maintainingthe homologously recombinant cell in vitro or in vivo under conditionsappropriate for expression of the gene.

The present invention further relates to a method of making apolypeptide of the present invention by altering the expression of atargeted endogenous gene in a cell in vitro or in vivo wherein the geneis not normally expressed in the cell, comprising the steps of: a)transfecting the cell in vitro with a polynucleotide construct, thepolynucleotide construct comprising: (i) a targeting sequence; (ii) aregulatory sequence and/or a coding sequence; and (iii) an unpairedsplice donor site, if necessary, thereby producing a transfected cell;(b) maintaining the transfected cell in vitro or in vivo underconditions appropriate for homologous recombination, thereby producing ahomologously recombinant cell; and c) maintaining the homologouslyrecombinant cell in vitro or in vivo under conditions appropriate forexpression of the gene thereby making the polypeptide.

The present invention further relates to a polynucleotide construct thatalters the expression of a targeted gene in a cell type in which thegene is not normally expressed. This occurs when a polynucleotideconstruct is inserted into the chromosomal DNA of the target cell,wherein the polynucleotide construct comprises: a) a targeting sequence;b) a regulatory sequence and/or coding sequence; and c) an unpairedsplice-donor site, if necessary. Further included are polynucleotideconstructs, as described above, wherein the construct further comprisesa polynucleotide that encodes a polypeptide and is in-frame with thetargeted endogenous gene after homologous recombination with chromosomalDNA.

The compositions may be produced, and methods performed, by techniquesknown in the art, such as those described in U.S. Pat. Nos: 6,054,288;6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965,125;5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734;International Publication Nos: WO96/29411, WO 94/12650; and scientificarticles described by Koller et al., (1994) Annu. Rev. Immunol.10:705-730; the disclosures of each of which are incorporated byreference in their entireties).

The OBG3 gene expression in mammalian, and typically human, cells may berendered defective, or alternatively it may be enhanced, with theinsertion of an OBG3 genomic or cDNA sequence with the replacement ofthe OBG3 gene counterpart in the genome of an animal cell by an OBG3polynucleotide according to the invention. These genetic alterations maybe generated by homologous recombination events using specific DNAconstructs that have been previously described.

One kind of host cell that may be used are mammalian zygotes, such asmurine zygotes. For example, murine zygotes may undergo microinjectionwith a purified DNA molecule of interest, for example a purified DNAmolecule that has previously been adjusted to a concentration range from1 ng/ml—for BAC inserts-3 ng/μl—for P1 bacteriophage inserts in 10 mMTris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine,and 70 μM spermidine. When the DNA to be microinjected has a large size,polyamines and high salt concentrations can be used in order to avoidmechanical breakage of this DNA, as described by Schedl et al ((1993)Nature 362(6417):258-61).

Any one of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells that provide the appropriatesignals to preserve this embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells are primary embryonicfibroblasts that are established from tissue of day 13-day 14 embryos ofvirtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (1993; Methods Enzymol 225:803-23) andare inhibited in growth by irradiation, such as described by Robertson((1987) Embryo-derived stem cell lines. In: E. J. Robertson Ed.Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRLPress, Oxford), or by the presence of an inhibitory concentration ofLIF, such as described by Pease and Williams (1990; Exp Cell Res190(2):209-11).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skilled artisan.

IV. Transgenic Animals

The present invention also provides methods and compositions for thegeneration of non-human animals and plants that express recombinant OBG3polypeptide, i.e. recombinant gOBG3 polypeptide fragment or full-lengthOBG3 polypeptide. The animals or plants can be transgenic, i.e. each oftheir cells contains a gene encoding the OBG3 polypeptide, or,alternatively, a polynucleotide encoding the polypeptide can beintroduced into somatic cells of the animal or plant, e.g. into mammarysecretory epithelial cells of a mammal. In preferred embodiments, thenon-human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.In further preferred embodiments, said gene encoding said gOBG3polypeptide fragment or said full-length OBG3 polypeptide comprises thepolynucleotide of SEQ ID NO:5.

Methods of making transgenic animals such as mammals are well known tothose of skill in the art, and any such method can be used in thepresent invention. Briefly, transgenic mammals can be produced, e.g., bytransfecting a pluripotential stem cell such as an ES cell with apolynucleotide encoding a polypeptide of interest. Successfullytransformed ES cells can then be introduced into an early stage embryothat is then implanted into the uterus of a mammal of the same species.In certain cases, the transformed (“transgenic”) cells will comprisepart of the germ line of the resulting animal, and adult animalscomprising the transgenic cells in the germ line can then be mated toother animals, thereby eventually producing a population of transgenicanimals that have the transgene in each of their cells, and which canstably transmit the transgene to each of their offspring. Other methodsof introducing the polynucleotide can be used, for example introducingthe polynucleotide encoding the polypeptide of interest into afertilized egg or early stage embryo via microinjection. Alternatively,the transgene may be introduced into an animal by infection of zygoteswith a retrovirus containing the transgene (Jaenisch, R. (1976) ProcNatl Acad Sci USA 73, 1260-1264). Methods of making transgenic mammalsare described, e.g., in Wall et al. (1992) J Cell Biochem 199249(2):113-20; Hogan, et al. (1986) in Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; in WO 91/08216; or in U.S. Pat. No. 4,736,866.

In a preferred method, the polynucleotides ares microinjected into thefertilized oocyte. Typically, fertilized oocytes are microinjected usingstandard techniques, and then cultured

in vitro until a “pre-implantation embryo” is obtained. Suchpre-implantation embryos preferably contain approximately 16 to 150cells. Methods for culturing fertilized oocytes to the pre-implantationstage are described, e.g., by Gordon et al. ((1984) Methods inEnzymology, 101, 414); Hogan et al. ((1986) in Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y) (for the mouse embryo); Hammer et al. ((1985)Nature, 315, 680) (for rabbit and porcine embryos); Gandolfi et al.((1987) J Reprod Fert 81, 23-28); Rexroad et al. ((1988) J Anim Sci 66,947-953) (for ovine embryos); and Eyestone et al. ((1989) J Reprod Fert85, 715-720); Camous et al. ((1984) J Reprod Fert 72, 779-785); andHeyman et al. ((1987) Theriogenology 27, 5968) (for bovine embryos); thedisclosures of each of which are incorporated herein in theirentireties. Pre-implantation embryos are then transferred to anappropriate female by standard methods to permit the birth of atransgenic or chimeric animal, depending upon the stage of developmentwhen the transgene is introduced.

As the frequency of transgene incorporation is often low, the detectionof transgene integration in pre-implantation embryos is often desirableusing any of the herein-described methods. Any of a number of methodscan be used to detect the presence of a transgene in a pre-implantationembryo. For example, one or more cells may be removed from thepre-implantation embryo, and the presence or absence of the transgene inthe removed cell or cells can be detected using any standard method e.g.PCR. Alternatively, the presence of a transgene can be detected in uteroor post partum using standard methods.

In a particularly preferred embodiment of the present invention,transgenic mammals are generated that secrete recombinant OBG3polypeptides in their milk. As the mammary gland is a highly efficientprotein-producing organ, such methods can be used to produce proteinconcentrations in the gram per liter range, and often significantlymore. Preferably, expression in the mammary gland is accomplished byoperably linking the polynucleotide encoding the OBG3 polypeptide to amammary gland specific promoter and, optionally, other regulatoryelements. Suitable promoters and other elements include, but are notlimited to, those derived from mammalian short and long WAP, alpha,beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN5′ genes,as well as the the mouse mammary tumor virus (MMTV) promoter. Suchpromoters and other elements may be derived from any mammal, including,but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guineapigs. Promoter and other regulatory sequences, vectors, and otherrelevant teachings are provided, e.g., by Clark (1998) J Mammary GlandBiol Neoplasia 3:337-50; Jost et al. (1999) Nat Biotechnol 17:160-4;U.S. Pat. Nos. 5,994,616; 6,140,552; 6,013,857; Sohn et al. (1999) DNACell Biol. 18:845-52; Kim et al. (1999) J Biochem (Japan) 126:320-5;Soulier et al. (1999) Euro J Biochem 260:533-9; Zhang et al. (1997) ChinJ Biotech 13:271-6; Rijnkels et al. (1998) Transgen Res 7:5-14; Korhonenet al. (1997) Euro J Biochem 245:482-9; Uusi-Oukari et al. (1997)Transgen Res 6:75-84; Hitchin et al. (1996) Prot Expr Purif 7:247-52;Platenburg et al. (1994) Transgen Res 3:99-108; Heng-Cherl et al. (1993)Animal Biotech 4:89-107; and Christa et a]. (2000) Euro J Biochem267:1665-71; the entire disclosures of each of which is hereinincorporated by reference.

In another embodiment, the polypeptides of the invention can be producedin milk by introducing polynucleotides encoding the polypeptides intosomatic cells of the mammary gland in vivo, e.g. mammary secretingepithelial cells. For example, plasmid DNA can be infused through thenipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens etal. (2000) Biochim. Biophys. Acta 1523:161-171), in association with aligand that can lead to receptor-mediated endocytosis of the construct(see, e.g., Sobolev et al. (1998) 273:7928-33), or in a viral vectorsuch as a retroviral vector, e.g. the Gibbon ape leukemia virus (see,e.g.,. Archer et al. (1994) PNAS 91:6840-6844). In any of theseembodiments, the polynucleotide may be operably linked to a mammarygland specific promoter, as described above, or, alternatively, anystrongly expressing promoter such as CMV or MoMLV LTR.

The suitability of any vector, promoter, regulatory element, etc. foruse in the present invention can be assessed beforehand by transfectingcells such as mammary epithelial cells, e.g. MacT cells (bovine mammaryepithelial cells) or GME cells (goat mammary epithelial cells), in vitroand assessing the efficiency of transfection and expression of thetransgene in the cells.

For in vivo administration, the polynucleotides can be administered inany suitable formulation, at any of a range of concentrations (e.g.1-500 μg/ml, preferably 50-100 μg/ml), at any volume (e.g. 1 -100 ml,preferably 1 to 20 ml), and can be administered any number of times(e.g. 1, 2, 3, 5, or 10 times), at any frequency (e.g. every 1, 2, 3, 5,10, or any number of days). Suitable concentrations, frequencies, modesof administration, etc. will depend upon the particular polynucleotide,vector, animal, etc., and can readily be determined by one of skill inthe art.

In a preferred embodiment, a retroviral vector such as as Gibbon apeleukemia viral vector is used, as described in Archer et al. ((1994)PNAS 91:6840-6844). As retroviral infection typically requires celldivision, cell division in the mammary glands can be stimulated inconjunction with the administration of the vector, e.g. using a factorsuch as estrodiol benzoate, progesterone, reserpine, or dexamethasone.Further, retroviral and other methods of infection can be facilitatedusing accessory compounds such as polybutene.

In any of the herein-described methods for obtaining OBG3 polypeptidesfrom milk the quantity of milk obtained, and thus the quantity of OBG3polypeptides produced, can be enhanced using any standard method oflacation induction, e.g. using hexestrol, estrogen, and/or progesterone.

The polynucleotides used in such embodiments can either encode afull-length OBG3 polypeptide or a gOBG3 polypeptide fragment. Typically,the encoded polypeptide will include a signal sequence to ensure thesecretion of the protein into the milk. Where a full-length OBG3sequence is used, the full-length protein can, e.g., be isolated frommilk and cleaved in vitro using a suitable protease. Alternatively, asecond, protease-encoding polynucleotide can be introduced into theanimal or into the mammary gland cells, whereby expression of theprotease results in the cleavage of the OBG3 polypeptide in vivo,thereby allowing the direct isolation of gOBG3 polypeptide fragmentsfrom milk.

V. Pharmaceutical or Physiologically Acceptable Compositions of theInvention

The gOBG3 polypeptide fragments of the invention can be administered tonon-human animals and/or humans, alone or in pharmaceutical orphysiologically acceptable compositions where they are mixed withsuitable carriers or excipient(s). The pharmaceutical or physiologicallyacceptable composition is then provided at a therapeutically effectivedose. A therapeutically effective dose refers to that amount of gOBG3fragment sufficient to result in prevention or amelioration of symptomsor physiological status of obesity-related diseases or disorders asdetermined by the methods described herein. A therapeutically effectivedose can also refer to the amount of gOBG3 fragment necessary for areduction in weight or a prevention of an increase in weight orprevention of an increase in the rate of weight gain in persons desiringthis affect for cosmetic reasons. A therapeutically effective dosage ofa gOBG3 fragment of the invention is that dosage that is adequate topromote weight loss or weight gain with continued periodic use oradministration. Techniques for formulation and administration of OBG3polypeptide fragments may be found in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., latest edition.

Other diseases or disorders that OBG3 polypeptide fragments of theinvention could be used to treat or prevent include, but are not limitedto, obesity and obesity-related diseases and disorders such as obesity,impaired glucose tolerance, insulin resistance, atherosclerosis,atheromatous disease, heart disease, hypertension, stroke, Syndrome X,Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) andInsulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).Diabetes-related complications to be treated by the methods of theinvention include microangiopathic lesions, ocular lesions, retinopathy,neuropathy, and renal lesions. Heart disease includes, but is notlimited to, cardiac insufficiency, coronary insufficiency, and highblood pressure. Other obesity-related disorders to be treated bycompounds of the invention include hyperlipidemia and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, cancer-related weight loss,anorexia, and bulimia. The gOBG3 polypeptide fragments may also be usedto enhance physical performance during work or exercise or enhance afeeling of general well-being. Physical performance activities includewalking, running, jumping, lifting and/or climbing.

The gOBG3 polypeptide fragments or antagonists thereof may also be usedto treat dyslexia, attention-deficit disorder (ADD),attention-deficit/hyperactivity disorder (ADHD), and psychiatricdisorders such as schizophrenia by modulating fatty acid metabolism,more specifically, the production of certain long-chain polyunsaturatedfatty acids.

It is expressly considered that the gOBG3 polypeptide fragments of theinvention may be provided alone or in combination with otherpharmaceutically or physiologically acceptable compounds. Othercompounds useful for the treatment of obesity and other diseases anddisorders are currently well-known in the art.

In a preferred embodiment, the gOBG3 polypeptide fragments are usefulfor, and used in, the treatment of insulin resistance and diabetes usingmethods described herein and known in the art. More particularly, apreferred embodiments relates to process for the therapeuticmodification and regulation of glucose metabolism in an animal or humansubject, which comprises administering to a subject in need of treatment(alternatively on a timed daily basis) an OBG or OBG3 polypeptidefragment (or polynucleotide encoding said polypeptide) in dosage amountand for a period sufficient to reduce plasma glucose levels in saidanimal or human subject.

Further preferred embodiments relate to methods for the prophylaxis ortreatment of diabetes comprising administering to a subject in need oftreatment (alternatively on a timed daily basis) an OBG or OBG3polypeptide fragment (or polynucleotide encoding said polypeptide) indosage amount and for a period sufficient to reduce plasma glucoselevels in said animal or human subject.

Routes of Administration.

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. A particularly useful methodof administering compounds for promoting weight loss involves surgicalimplantation, for example into the abdominal cavity of the recipient, ofa device for delivering OBG3or gOBG3 polypeptide fragments over anextended period of time. Other particularly preferred routes ofadministration are aerosol and depot formulation. Sustained releaseformulations, particularly depot, of the invented medicaments areexpressly contemplated.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable acceptable carrier and atleast one polypeptide that is an OBG3 polypeptide fragment of theinvention. For injection, the agents of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer such as a phosphate or bicarbonate buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.gelatin, for use in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection maybe presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization maybe employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage.

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to increase leptin orlipoprotein uptake or binding in an in vitro system. Such informationcan be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50, (the dose lethal to 50% of the testpopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio between LD50and ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to maintain orprevent weight loss or gain, depending on the particular situation.Dosages necessary to achieve these effects will depend on individualcharacteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-90%; andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

A preferred dosage range for the amount of an OBG3 polypeptide fragmentof the invention, which can be administered on a daily or regular basisto achieve desired results, including a reduction in levels ofcirculating plasma triglyceride-rich lipoproteins, range from 0.01-0.5mg/kg body mass. A more preferred dosage range is from 0.05 -0.1 mg/kg.Of course, these daily dosages can be delivered or administered in smallamounts periodically during the course of a day. It is noted that thesedosage ranges are only preferred ranges and are not meant to be limitingto the invention.

VI. Methods of Treatment

Treatment of mice with gOBG3 polypeptide fragments results in decreasedtriglyceride levels, decreased free fatty acid levels, decreased glucoselevels, and decreased body weight as well as increased muscle oxidation.

The invention is drawn inter alia to methods of preventing or treatingobesity-related diseases and disorders comprising providing anindividual in need of such treatment with a gOBG3 polypeptide fragmentof the invention. Preferably, the OBG3 polypeptide fragment hasobesity-related activity either in vitro or in vivo. Preferably the OBG3polypeptide fragment is provided to the individual in a pharmaceuticalcomposition that is preferably taken orally. Preferably the individualis a mammal, and most preferably a human. In preferred embodiments, theobesity-related disease or disorder is selected from the groupconsisting of atherosclerosis, cardiovascular disease, impaired glucosetolerance, insulin resistance, hypertension, stroke, Syndrome X, Type Idiabetes, Type II diabetes and lipoatrophic diabetes. Diabetes-relatedcomplications to be treated by the methods of the invention includemicroangiopathic lesions, ocular lesions, retinopathy, neuropathy andrenal lesions. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Otherobesity-related disorders to be treated by compounds of the inventioninclude hyperlipidemia, hypertriglyceridemia, and hyperuricemia. Yetother obesity-related diseases or disorders of the invention includecachexia, wasting, AIDS-related weight loss, neoplasia-related weightloss, anorexia, and bulimia. In highly preferred embodiments, OBG3polypeptide polypeptide fragments in pharmaceutical compositions areused to modulate body weight in healthy individuals for cosmeticreasons.

The invention also features a method of preventing or treatingobesity-related diseases and disorders comprising providing anindividual in need of such treatment with a compound identified byassays of the invention (described in Section VI of the PreferredEmbodiments of the Invention and in the Examples). Preferably thesecompounds antagonize or agonize effects of gOBG3 polypeptide fragmentsin cells in vitro, muscles ex vivo, or in animal models. Alternatively,these compounds agonize or antagonize the effects of gOBG3 polypeptidefragments on leptin and/or lipoprotein uptake and/or binding.Optionally, these compounds prevent the interaction, binding, or uptakeof gOBG3 polypeptide fragments with LSR in vitro or in vivo. Preferably,the compound is provided to the individual in a pharmaceuticalcomposition that is preferably taken orally. Preferably the individualis a mammal, and most preferably a human. In preferred embodiments, theobesity-related disease or disorder is selected from the groupconsisting of obesity and obesity-related diseases and disorders such asatherosclerosis, heart disease, impaired glucose tolerance, insulinresistance, hypertension, stroke, Syndrome X, Type I diabetes, Type IIdiabetes, and lipoatrophic diabetes. Diabetes-related complications tobe treated by the methods of the invention include microangiopathiclesions, ocular lesions, retinopathy, neuropathy and renal lesions.Heart disease includes, but is not limited to, cardiac insufficiency,coronary insufficiency, and high blood pressure. Other obesity-relateddisorders to be treated by compounds of the invention includehyperlipidemia, hypertriglyceridemia, and hyperuricemia. Yet otherobesity-related diseases or disorders of the invention include cachexia,wasting, AIDS-related weight loss, neoplasia-related weight loss,anorexia, and bulimia. In highly preferred embodiments, thepharmaceutical compositions are used to modulate body weight forcosmetic reasons.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type I diabetes, Type II diabetes, or insulin resistance, incombination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control blood glucose in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance,alone, without combination of insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to control body weight in some individuals, particularly thosewith Type II diabetes or insulin resistance, alone, without combinationof insulin therapy.

In a further preferred embodiment, the present invention may be used incomplementary therapy, particularly in some individuals, particularlythose with Type I diabetes, Type II diabetes, or insulin resistance, toimprove their weight or glucose control in combination with an oralinsulin secretagogue or an insulin sensitising agent. Preferably, theoral insulin secretagogue is 1,1-dimethyl-2-(2-morpholinophenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected fromtolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride,glipizide and glidazide. Preferably, the insulin sensitising agent isselected from metformin, ciglitazone, troglitazone and pioglitazone.

The present invention further provides a method of improving the bodyweight or glucose control of some individuals, particularly those withType I diabetes, Type II diabetes, or insulin resistance, alone, withoutan oral insulin secretagogue or an insulin sensitising agent.

In a further preferred embodiment, the present invention may beadministered either concomitantly or concurrently, with the oral insulinsecretagogue or insulin sensitising agent for example in the form ofseparate dosage units to be used simultaneously, separately orsequentially (either before or after the secretagogue or either beforeor after the sensitising agent). Accordingly, the present inventionfurther provides for a composition of pharmaceutical or physiologicallyacceptable composition and an oral insulin secretagogue or insulinsensitising agent as a combined preparation for simultaneous, separateor sequential use for the improvement of body weight or glucose controlin some individuals, particularly those with Type I diabetes, Type IIdiabetes, or insulin resistance.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an insulin sensitiser.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type I diabetes, Type II diabetes, or insulinresistance, in combination with insulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition can be used asa method to improve insulin sensitivity in some individuals,particularly those with Type II diabetes or insulin resistance, withoutinsulin therapy.

In further preferred embodiments, the present invention of saidpharmaceutical or physiologically acceptable composition furtherprovides a method for the use as an inhibitor of the progression fromimpaired glucose tolerance to insulin resistance.

More generally, the instant invention is drawn to treatment with gOBG3polypeptide fragments where an individual is shown to have a particulargenotype for an APM1 marker (APM1 designates the human homolog of thefull-length OBG3 polypeptide), or where they have been shown to have areduced amount of plasma APM1, either full-length or preferably a morebiologically active fragment of APM1, as compared to control values,e.g. values representative of non-diseased individuals, or as comparedto that individual prior to the onset of a disease or condition. Ineither case, treatment comprises providing pharmaceutically acceptablegOBG3 or OBG3 polypeptide fragments to the individual. The exact amountof gOBG3 fragment provided would be determined through clinical trialsunder the guidance of qualified physicians, but would be expected to bein the range of 5-7 mg per individual per day. In general, a preferredrange would be from 0.5 to 14 mg per individual per day, with a highlypreferred range being between 1 and 10 mg per individual per day.Individuals who could benefit from treatment with gOBG3 or OBG3polypeptide fragments could be identified through at least two methods:plasma serum level determinations and genotyping.

OBG3/APM1 Levels

Preliminary studies have shown that obese people have lower levels offull-length OBG3/APM1 than non-obese people. The invention envisionstreatment of individuals (preferably obese) that have low levels offull-length OBG3/APM1 with gOBG3 polypeptide fragments of the invention.In addition, the invention preferably is drawn to treatment ofindividuals with low levels of the biologically active fragment ofOBG3/APM1 with goBG3 polypeptide fragments of the invention. In furtherembodiments, OBG3 or OBG3 polypeptide fragments of the present inventionare administered to individuals, preferably obese individuals, thatlevels of full-length OBG3 (or alternatively a mature OBG3 polypeptidefragment) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, about100% or 100% lower than non-obese individuals, preferably healthyindividuals as determined by a physician using normal standards in theart. Methods to determine and compare the levels of full-ength OBG3 inindividuals are well-known in the art and include, but are not limitedto using an antibody specific for APMI in a format such as a RadioImmune Assay, ELISA, Western blot, dotblot, or as part of an array, forexample. Methods of generating antibodies to, and detection of, APM1 andfragments thereof as well as to proteins with SNPs are included in thepresent invention and are discussed in PCT/IB99/01858, U.S. applicationSer. No. 09/434,848, and WO 99/07736, hereby incorporated herein byreference in its entirety including and drawings, figures, or tables.Further, antibodies specific for OBG3/gOBG3 polypeptide fragments of theinvention, their generation, and their use are described herein.

VII. Assays for Identifying Modulators of OBG3 Polypeptide FragmentActivity

The invention features methods of screening for one or more compoundsthat modulate gOBG3 polypeptide fragment activity in cells, thatincludes providing potential compounds to be tested to the cells, andwhere modulation of a gOBG3 polypeptide fragment effect or activityindicates the one or more compounds. Exemplary assays that may be usedare described in the Examples 4-5, 7-14, 16, and 18. To these assayswould be added compounds to be tested for their inhibitory orstimulatory activity as compared to the effects of gOBG3 polypeptidefragment alone. Other assays in which an effect is observed based on theaddition of gOBG3 polypeptide fragment can also be used to screen formodulators of gOBG3 polypeptide fragment activity or effects of thepresence of gOBG3 polypeptide fragment on cells. The essential step isto apply an unknown compound and then to monitor an assay for a changefrom what is seen when only gOBG3 polypeptide fragment is applied to thecell. A change is defined as something that is significantly differentin the presence of the compound plus gOBG3 polypeptide fragment comparedto gOBG3 polypeptide fragment alone. In this case, significantlydifferent would be an “increase” or a “decrease” in a measurable effectof at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.

The term “modulation” as used herein refers to a measurable change in anactivity. Examples include, but are not limited to, lipolysis stimulatedreceptor (LSR) modulation, leptin modulation, lipoprotein modulation,plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight.These effects can be in vitro or preferably in vivo. Modulation of anactivity can be either an increase or a decrease in the activity. Thus,LSR activity can be increased or decreased, leptin activity can beincreased or decreased, and lipoprotein activity can be increased ordecreased. Similarly, FFA, TG, and glucose levels (and weight) can beincreased or decreased in vivo Free Fatty Acid oxidation can beincreased or decreased in vivo or ex vivo.

By “LSR” activity is meant expression of LSR on the surface of the cell,or in a particular conformation, as well as its ability to bind, uptake,and degrade leptin and lipoprotein. By “leptin” activity is meant itsbinding, uptake and degradation by LSR, as well as its transport acrossa blood brain barrier, and potentially these occurrences where LSR isnot necessarily the mediating factor or the only mediating factor.Similarly, by “lipoprotein” activity is meant its binding, uptake anddegradation by LSR, as well as these occurrences where LSR is notnecessarily the mediating factor or the only mediating factor. Exemplaryassays are provided in Example 4-5, 7-14, 16, and 18. These assay andother comparable assays can be used to determine/identify compounds thatmodulate gOBG3 polypeptide fragment activity. In some cases it may beimportant to identify compounds that modulate some but not all of thegOBG3 polypeptide fragment activities, although preferably allactivities are modified.

The term “increasing” as used herein refers to the ability of a compoundto increase an gOBG3 polypeptide fragment activity in some measurableway compared to the effect of an gOBG3 polypeptide fragment in itsabsence. As a result of the presence of the compound leptin bindingand/or uptake might increase, for example, as compared to controls inthe presence of the gOBG3 polypeptide fragment alone. Preferably, anincrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, or 75% compared to the level of activity in the presence ofthe gOBG3 polypeptide fragment.

Similarly, the term “decreasing” as used herein refers to the ability ofa compound to decrease an activity in some measurable-way compared tothe effect of a gOBG3 fragment in its absence. For example, the presenceof the compound decreases the plasma concentrations of FFA, TG, andglucose in mice. Also as a result of the presence of a compound leptinbinding and/or uptake might decrease, for example, as compared tocontrols in the presence of the gOBG3 polypeptide fragment alone.Preferably, a decrease in activity is at least 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity inthe presence of the gOBG3 polypeptide fragment alone.

The invention features a method for identifying a potential compound tomodulate body mass in individuals in need of modulating body masscomprising: a) contacting a cell with a gOBG3 polypeptide fragment and acandidate compound; b) detecting a result selected from the groupconsisting of LSR modulation, leptin modulation, lipoprotein modulation,FFA oxidation modulation; and c) wherein said result identifies saidpotential compound if said result differs from said result when saidcell is contacted with the gOBG3 polypeptide fragment alone.

In preferred embodiments, said contacting further comprises a ligand ofsaid LSR. Preferably said ligand is selected from the group consistingof cytokine, lipoprotein, free fatty acids, and C1q, and more preferablysaid cytokine is leptin, and most preferably said leptin is a leptinpolypeptide fragment as described in U.S. Provisional application No.60/155,506 hereby incorporated by reference herein in its entiretyincluding any figures, drawings, or tables.

In other preferred embodiments, said gOBG3 polypeptide fragment is mouseor is human. In other preferred embodiments, said cell is selected fromthe group consisting of PLC, CHO-K1, Hep3B, and HepG2.

In yet other preferred embodiments, said lipoprotein modulation isselected from the group consisting of binding, uptake, and degradation.Preferably, said modulation is an increase in said binding, uptake, ordegradation. Alternatively, said modulation is a decrease in saidbinding, uptake, or degradation.

In other preferred embodiments, leptin modulation is selected from thegroup consisting of binding, uptake, degradation, and transport.Preferably, said modulation is an increase in said binding, uptake,degradation, or transport. Alternatively, said modulation is a decreasein said binding, uptake, degradation, or transport. Preferably, saidtransport is across a blood-brain barrier.

In yet other preferred embodiments, said LSR modulation is expression onthe surface of said cell. Preferably, said detecting comprises FACS,more preferably said detecting further comprises antibodies that bindspecifically to said LSR, and most preferably said antibodies bindspecifically to the carboxy terminus of said LSR.

In still other preferred embodiments, said potential compound isselected from the group consisting of peptides, peptide libraries,non-peptide libraries, peptides, fatty acids, lipoproteins, medicaments,antibodies, small molecules, and proteases. Other characteristics andadvantages of the invention are described in the Brief Description ofthe Figures and the Examples. These are meant to be exemplary only, andnot to limit the invention in any way. Throughout this application,various publications, patents and published patent applications arecited. The disclosures of these publications, patents and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure.

VIII: Assays for Identifying Antagonists of Homotrimeric gOBG3Polypeptide Fragment Activity

The invention features methods of screening compounds for one or moreantagonists of homotrimeric gOBG3 polypeptide fragment activity, whereinsaid activity is selected from but not restricted to weight reduction,lipid partitioning, lipid metabolism, and insulin-like activity.Preferred said compound is selected from but is not restricted to smallmolecular weight organic or inorganic compound, protein, peptide,carbohydrate, or lipid. Preferred said gOBG3 polypeptide fragmentforming homotrimers having said activity is selected from amino acids18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244,27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244,36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2wherein the cysteine at position 36 is substituted with an amino acidother than cysteine. Other preferred said gOBG3 polypeptide fragmentforming homotrimers having said activity is selected from amino acids18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247,27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247,36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247,or 45-247 of SEQ ID NO:4, wherein the cysteine at position 39 issubstituted with an amino acid other than cysteine. Other preferred saidgOBG3 polypeptide fragment forming homotrimers having said activity isselected from amino acids 37-244, 38-244, 39-244, 40-244, 41-244, or42-244 of SEQ ID NO:2. Other preferred said gOBG3 polypeptide fragmentforming homotrimers having said activity is selected from amino acids40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4.

The invention further features methods of screening compounds for saidantagonist of gOBG3 polypeptide fragment activity comprising: a)contacting said homotrimeric gOBG3 polypeptide fragment with or withoutsaid compound; b) detecting a result on the basis of activity, whereinsaid activity is selected from but not restricted to lipid partitioning,lipid metabolism, and insulin-like activity; and c) wherein said resultidentifies said compound as an antagonist of homotrimeric gOBG3polypeptide fragment activity if said result with compound differs fromsaid result without compound. Exemplary assays that may be used aredescribed in Examples 4 and 18.

EXAMPLES

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein allof which form part of the instant invention.

It should be noted that the term full-length OBG3 polypeptide usedthroughout the specification is intended to encompass the proteinhomologs mouse ACRP30 [Scherer, et al., “A novel serum protein similarto C1q, produced exclusively in adipocytes”; J Biol Chem 270,26746-26749 (1995)], mouse AdipoQ [Hu, et al., “AdipoQ is a noveladipose-specific gene dysregulated in obesity”, J Biol Chem 271,10697-10703 (1996)], human APM1 [Maeda, et al., “cDNA cloning andexpression of a novel adipose specific collagen-like factor, APM1(AdiPose Most abundant Gene transcript 1)”, Biochem Biophys Res Commun221, 286-289 (1996)) and human GBP28 [Nakano, et al., “Isolation andcharacterization of GBP28, a novel gelatin-binding protein purified fromhuman plasma”, J Biochem (Tokyo) 120, 803-812 (1996)]. OBG3 is alsointended to encompass other homologs. gOBG3 is understood to refer topolypeptide fragments of full-length OBG3 polypeptide comprised of theglobular domain. Example 1

Production of Recombinant OBG3

An exemplary method for generating recombinant OBG3 is given below.Although the method describes the production of the mouse analog, aperson with skill in the art would be able to use the informationprovided to produce other OBG3 analogs, including but not limited to thehuman analog. An alignment of the amino acid sequences of the human(APM1) and mouse (AdipoQ and ACRP30) OBG3 is shown in FIG. 1. Therecombinant OBG3 analog is cloned in pTRC His B (Invitrogen)between-BamH1 and Xho1 (FIG. 2) and maintained in E. coli DH5-alpha. Thesequence of the OBG3 insert corresponds to ACRP30 genbank U37222 bases88 to 791 except in position 382 where in #3 G replaces A found in ACRP30 (V instead of M). The corresponding nucleotide in AdipoQ U49915 is Gas in clone #3. The amino acid V is also conserved in the human sequenceAPM1 D45371.

Culture:

Plate out bacteria in LB agar media containing 100 μg/mL ampicillin.Inoculate 1 colony into 5 mL media (no agar) at 37° C. overnight. Add 2mL of this initial culture into 500 mL Erlenmeyer flasks containing 200mL LB media and 100 μg/mL ampicillin. Incubate at 37° C. in an orbitalshaker until the OD₆₀₀=0.2. Add IPTG to a final concentration of 1 mM(stock solution=1 M). Incubate at 37° C. overnight.

Lysis:

Pellet the bacteria by centrifugation (Sorvall, 3500 rpm, 15 min, 4° C.)in a pre-weighed tube.

At 4° C. resuspend the pellet in 3 mL/g of lysis buffer

Add 40 μL/g PMSF 10 mM

Add 80 μL/g of lysozyme 10 mg/mL

Incubate 20 min on ice, shaking intermittently

Add 30 μL/g 10% sodium deoxycholate

Incubate at 37° C. until the lysate is viscous

Freeze in liquid Nitrogen and thaw at 37° C. three times

Sonicate 2×, 30 sec, 25% cycle, 2.5 power level

Centrifuge 30 min, 15000 rpm, 4° C.

Recover the supernatant

Note: The lysate can be stored frozen before or after the sonicationstep.

Batch Purification:

1. Pack 1 mL of Probond resin (Invitrogen; 1 mL=2 mL suspended gel) intoa 5 mL column. Wash with 5 mL PBS.

2. Apply 5 mL bacterial supernatant to the 1 mL of gel. (If volume isvery high, use several small columns.)

3. Wash with 24 mL phosphate buffer, pH 7.8, followed by a wash with 24mL phosphate buffer, pH 6.

4. Elute with imidazole buffer and collect fractions of 1 mL.

5. Analyze fractions by OD at 280 nm or by SDS-PAGE (12.5%; dilution ½in 2× sample buffer) under reducing conditions (100° C., 5 min)

6. Pool the fractions containing protein (usually fraction numbers 2-4for concentrations of 0.8-1 mg/mL and fractions 1, 5 and 6 forconcentrations of 0.2-0.4 mg/mL).

7. Dialyze thoroughly against 1×PBS, 24 mM ammonium bicarbonate or 50 mMTris, pH 7.4 containing 250 nM NaCl. Concentrate by Speed-Vac if needed.

8. Analyze protein by the Lowry method.

9. Aliquot and store at −20° C.

Purification on Liquid Chromatography System

1. Pack 5 mL of Probond resin into a 5 mL column.

2. Wash with 4 bed volumes of phosphate buffer pH 7.8, 1 mL/min.

3. Inject 25 mL lysate (filtered on 0.45μ or centrifuged at 3000 rpm, 30min, 4° C., Beckman Allegra 6R) at 0.5 mL/min.

4. Wash with 4 bed volumes of phosphate buffer, pH 7.8 at 1 mL/min.

5. Wash with 12 bed volumes of phosphate buffer pH 5.5 at 1 mL/min.

6. Elute bound fraction with phosphate buffer, pH 5.5, containing 1 Mimidazole at 1 mL/min.

7. Collect fractions, dialyze and analyze protein as described for batchpurification, steps 7-9.

Example 2 Generation of Globular OBG3 by Enzymatic Cleavage

Incubate purified OBG3 (obtained as described above or throughequivalent method) with acetylated Trypsin-Type V-S from Bovine Pancreas(Sigma E.C.=3.4.21.4 ) at 400 u/mg protein at 25° C. for 10 min.

Stop reaction by running the sample over a Poly-Prep Column (Biorad731-1550) at +4° C. containing immobilized Trypsin inhibitor.

Collect 1.0 mL fractions. Deternine protein concentration.

Pool the protein containing fractions and dialyze extensively againstPBS using dialysis tubing with M. W. cutoff=10,000 da.

Concentrate on Amicon YM-10 Centricon Filter (Millipore, M.W.cutoff=10,000 da). Sterile filter.

Determine final protein concentration using Markwell's modified Lowryprocedure (1981) or BCA protein assay (Pierce Chemical Co, Rockford,Ill.) and BSA as standard.

Check purity and efficiency of cleavage by SDS—PAGE analysis using a4-20% gradient gel. The intact OBG3 migrates as a single band atapproximately 37 kDa apparently due to co-transcribed vector sequencesattached to the histidine tag at the N-terminus of AdipoQ, and forms adimer at 74 kDa. The cleaved OBG3 forms a band at approx. 18 kDa(gOBG3). Additional degradation products, all smaller than 10 kDa arealso generated from the N-terminal region. These are separated from thedesired 18 kDa band by dialysis with semipermeable membranes with a MWcutoff of 10,000. The two potential cleavage sites for gOBG3 are shownin FIG. 3. The actual cleavage site has been identified as the one afteramino acid 103 (amino acid 100 for huma gOBG3 or APM1) (FIG. 7). Thatis, the N-terminus of the gOBG3 cleavage product is Lys 104 (Lys 101 forhuma gOBG3 or APM1).

Other enzymatic/proteolytic methods can also be used that yield apreferred gOBG3 polypeptide fragment, e.g. clostripain, adipsin,plasmin, collagenase, matrix metalloproteinase-1 (MMP-1), orprecerebellin processing protease. Other preferred enzymes wouldpreferably cleave OBG3 at a site close to the junction between thecollagen-like tail and the globular head (about amino acid 108 for humangOBG3 and about amino acid 111 for murine gOBG3), preferably permit thereaction to be easily stopped, preferably be easily removed using animmobilized inhibitor, or similar method, and preferably cuts theN-terminal fragment into small pieces (less than 10,000 MW). Thecleavage preferably results in the presence of no more than 8 collagenrepeats, more preferably no more than 3 collagen repeats, and mostpreferably no collagen repeats. A collagen repeat consists of Gly-X-Y. Adetermination of whether an active gOBG3 has been generated can bechecked using the in vitro and in vivo assays described herein (Examples4-6, 8-10).

Example 3 Generation of gOBG3 by Recombinant Methodology RestrictionSite Cloning

A first approach is to look for unique restriction sites near thebeginning of the globular head region (nucleic acid sequences of mouseand human OBG3 polypeptides are provided in the sequence listing). Ifpresent, it can be used to cleave within the 5′ collagen-like region andgenerate a C-terminal fragment comprised of the globular head region. Ifa unique site is not present, it is also possible, although moredifficult, to do this using restriction enzymes that cut in more thanone location by doing partial digestions. The 3′ end of the globularhead can be cut from its vector backbone using an appropriate enzyme.The globular head can then be cloned into an expression vector andconstructs containing the correct fragments can be identified. ForAdipoQ, Tau I seems to be a unique enzyme that would separate thecollagen tail from the globular head.

PCR Cloning

Another approach is to PCR the region of interest from the intactsequence (if cDNA is available) using primers with restriction sites onthe end so that PCR products can be directly cloned into vectors ofinterest. Alternatively, gOBG3 can also be generated using RT-PCR toisolate it from adipose tissue RNA.

E. coli Vector

For example, the AdipoQ globular domain can be cloned into pTrcHisB, byputting a Bam HI site on the sense oligo and a Xho I site on theantisense oligo. This allows isolation of the PCR product, digestion ofthat product, and ligation into the pTrcHisB vector that has also beendigested with Bam HI and Xho I (FIG. 4). The vector, pTrcHisB, has anN-terminal 6-Histidine tag, that allows purification of the overexpressed protein from the lysate using a Nickel resin column. ThepTrcHisB vector is used for over-expression of proteins in E. coli.

Exemplary oligos for cloning into the E. coli vector include:

A) OBG3 sense CTTAGTGGATCCCGCTTATGTGTATCGCTCAG 6 base pairs from theleft there is a 6 bp BamHI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

B) OBG3 antisense GCTGTFCTCGAGTCAGTTGGTATCATGG 6 base pairs from theleft there is a 6 bp. XhoI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

The following are exemplary PCR conditions.

Final concentrations in the reaction are:

1×PE Biosystems buffer A

1.5 mM MgCl₂

200 uM of each dNTP (dATP, dCTP, dGTP, dTTP)

2.5 Units of Amplitaq Gold from PE Biosystems

0.4 uM of each primer (sense and antisense)

10 ng of plasmid template

Cycling parameters:

95° C. 10min—1 cycle

95° C. 30sec

56° C. 30 sec

72° C. 30 sec

repeat above 3 steps for 30 cycles

72° C. 7 min—1 cycle.

BAC Vector

The globular head can also be over expressed in a Baculovirus systemusing the 6×His Baculovirus kit (Pharmingen), for example. The AdipoQglobular domain is cloned into the appropriate vector using enzymesavailable in the multiple cloning site. This allows over-expression ofthe protein in a eukaryotic system which has some advantages over the E.coli system, including: Multiple gene expression, Signal peptidecleavage, Intron splicing, Nuclear transport, Functional protein,Phosphorylation, Glycosylation, and Acylation.

Exemplary oligos for cloning into the Baculovirus vector are thefollowing:

A). OBG3 sense CTTAGTGAATTCGCTrATGTGTATCGCTCAGA 6 base pairs from theleft there is a 6 bp. EcoRI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

B). OBG3 antisense GCTGTTCTGCAGTCAGTTGGTATCATGG 6 base pairs from theleft there is a 6 bp. PstI site. Thus the region that is homologous tothe gene begins at nucleotide 13.

The following are exemplary PCR conditions.

Final concentrations in the reaction are:

1×PE Biosystems buffer A

1.5 mM MgCl₂

200 uM of each dNTP (dATP, dCTP, dGTP, dTTP)

2.5 Units of Amplitaq Gold from PE Biosystems

0.4 uM of each primer (sense and antisense)

10 ng of plasmid template

Cycling parameters:

95° C. 10min—1 cycle

95° C. 30 sec

60° C. 30 sec

72° C. 30 sec

repeat above 3 steps for 30 cycles

72° C. 7 min—1 cycle.

Mammalian Vector

Globular OBG3 can also be cloned into a mammalian expression vector andexpressed in and purified from mammalian cells, for example 3T3-L1 cells(undifferentiated adipocyte precursors). The globular head is thengenerated in an environment very close to its endogenous environment.However, this is not necessarily the most efficient way to make protein.

Example 4 In Vitro Tests of Obesity-Related Activity

The activity of various preparations and various sequence variants ofgOBG3 polypeptide fragments are assessed using various in vitro assaysincluding those provided below. These assays are also exemplary of thosethat can be used to develop gOBG3 polypeptide fragment antagonists andagonists. To do that, the effect of goBG3 polypeptide fragments in theabove assays, e.g. on leptin and/or LSR activity, in the presence of thecandidate molecules would be compared with the effect of gOBG3polypeptide fragments in the assays in the absence of the candidatemolecules. Since gOBG3 polypeptide fragments have been shown to reducebody,weight in mice on a high-cafeteria diet (Example 5), these assaysalso serve to identify candidate treatments for reducing (or increasing)body weight.

Liver Cell Line:

Tests of efficacy of gOBG3 polypeptide fragments on LSR can be performedusing liver cell lines, including for example, PLC, HepG2, Hep3B(human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat). Forhuman cell lines, APM1 and globular APM1 would be used preferentially;for rodents, full-length and globular AdipoQ/ACRP30 would be usedpreferentially.

BPRCL mouse liver cells (ATCC Repository) were plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media was changed on day 2. On day 3, the confluent monolayers werewashed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).Cells were incubated at 37° C. for 30 min with increasing concentrationsof recombinant AdipoQ (AQ) or globular AdipoQ (AQ-GH) in DMEM containing0.2% (w/v) BSA, 5 mM Hepes, 2 nM CaCl₂, 3.7 g/L sodium bicarbonate, pH7.5. Incubations were continued for 3 h at 37° C. after addition of 10ng/mL ¹²⁵I-mouse leptin (specific activity, 22100 cpm/ng). Monolayerswere washed 2 times consecutively with PBS containing 0.2% BSA, followedby 1 wash with PBS/BSA, and then 2 times consecutively with PBS. Cellswere lysed with 0.1 N NaOH containing 0.24 mM EDTA. Lysates werecollected into tubes, and counted in a gamma-counter. Results of anexemplary experiment are shown as the mean of triplicate determinationsin FIG. 5.

The results indicate that gOBG3 polypeptide fragments are at least 30%more efficient than OBG3 in increasing leptin uptake in a liver cellline (FIG. 5). This assay could be used to determine the efficiency ofgOBG3 polypeptide fragments and related compounds (or agonists orantagonists) to increase or decrease leptin uptake into the liver, aswell as the mechanism by which the gOBG3 polypeptide fragment/compoundexerts this effect.

Blood Brain Barrier Model:

The effect of gOBG3 polypeptide fragments on leptin transport in thebrain can be determined using brain-derived cells. One method that isenvisioned is to use the blood/brain barrier model described by Dehouck,et al., “An easier, reproducible, and mass-production method to studythe blood-brain barrier in vitro”, J Neurochem 54, 1798-1801 (1990);hereby incorporated herein by reference in its entirety (including anyfigures, tables, or drawings) that uses a co-culture of brain capillaryendothelial cells and astrocytes to test the effects of gOBG3polypeptide fragments on leptin (or other molecules) transport via LSRor other receptors.

This assay would be an indicator of the potential effect of gOBG3polypeptide fragments on leptin transport to the brain and could be usedto screen gOBG3 polypeptide fragment variants for their ability tomodulate leptin transport through LSR or other receptors in the brain.In addition, putative agonists and antagonists of the effect of gOBG3polypeptide fragments on leptin transport through LSR or other receptorscould also be screened using this assay. Increased transport of leptinacross the blood/brain barrier would presumably increase its action as asatiety factor.

FACS Analysis of LSR Expression

The effect of gOBG3 polypeptide fragments on LSR can also be determinedby measuring the level of LSR expression at the cell surface by flowsurface cytometry, using anti-LSR antibodies and fluorescent secondaryantibodies. Flow cytometry is a laser-based technology that is used tomeasure characteristics of biological particles. The underlyingprinciple of flow cytometry is that light is scattered and fluorescenceis emitted as light from the excitation source strikes the movingparticles.

This is a high through-put assay that could be easily adapted to screengOBG3 polypeptide fragments and variants as well as putative agonists orantagonists of gOBG3 polypeptide fragments. Two assays are providedbelow. The antibody, cell-line and goBG3 polypeptide fragment analogwould vary depending on the experiment, but a human cell-line, humananti-LSR antibody and globular APM1 could be used to screen forvariants, agonists, and antagonists to be used to treat humans.

Assay 1:

Cells are pretreated with either intact gOBG3 polypeptide fragments (oruntreated) before harvesting and analysis by FACS. Cells are harvestedusing non-enzymatic dissociation solution (Sigma), and then areincubated for 1 h at 4° C. with a 1:200 dilution of anti-LSR 81B or anirrelevant anti-serum in PBS containing 1% (w/v) BSA. After washingtwice with the same buffer, goat anti-rabbit FITC-conjugated antibody(Rockland, Gilbertsville, Pa.) is added to the cells, followed by afurther incubation for 30 min at 4° C. After washing, the cells arefixed in 2% formalin. Flow cytometry analysis is done on a FACSCaliburcytometer (Becton-Dickinson, Franklin Lakes, N.J.). The in vitro LiverCell Line assay (described above) has shown that LSR activity (leptinbinding) increases with increasing concentrations of gOBG3 polypeptidefragments. Whle not wishing to be bound by any particular theory, thiscould either be the result of an increased number of LSR binding siteson the cell surface, or a change in affinity for leptin. The FACS assaywould presumably be detecting changes in the number of LSR bindingsites, although changes in conformation reflecting changes in affinitymight also be detected. Preferably the antibody would be to theC-terminus of LSR.

Assay 2:

Cells are cultured in T175 flasks according to manufacturer'sinstructions for 48 hours prior to analysis.

Cells are washed once with FACS buffer (1×PBS/2% FBS, filtersterilized), and manually scraped from the flask in 10 mLs of FACSbuffer. The cell suspension is transferred to a 15 mL conical tube andcentrifuged at 1200 rpm, 4° C. for 5 minutes. Supernatant is discardedand cells are resuspended in 10 mL FACS buffer chilled to 4° C. A cellcount is performed and the cell density adjusted with FACS buffer to aconcentration of 1×10⁶ cells/mL. One milliliter of cell suspension wasadded to each well of a 48 well plate for analysis. Cells arecentrifuged at 1200 rpm for 5 minutes at 4° C. Plates are checked toensure that cells are pelleted, the supernatant is removed and cellsresuspended by running plate over a vortex mixer. One milliliter of FACSbuffer is added to each well, followed by centrifugation at 1200 rpm for5 minutes at 4° C. This described cell washing was performed a total of3 times.

Primary antibody, titered in screening experiments to determine properworking dilutions (for example 1:25, 1:50, 1:100, 1:200, 1:400,1:500,1:800, 1:1000, 1:2000, 1:4000, 1:5000, or 1:10000), is added tocells in a total volume of 50 μL FACS buffer. Plates are incubated for 1h at 4° C. protected from light. Following incubation, cells are washed3 times as directed above. Appropriate secondary antibody, titered inscreening experiments to determine proper working dilutions (for example1:25, 1:50, 1:100, 1:200, 1:400, 1:500, 1:800, 1:1000, 1:2000, 1:4000,1:5000, or 1:10000), is added to cells in a total volume of 50 μL FACSbuffer. Plates are incubated for 1 h at 4° C. protected from light.Following incubation, cells are washed 3 times as directed above. Uponfinal wash, cells are resuspended in 500 μL FACS buffer and transferedto a FACS acquisition tube. Samples are placed on ice protected fromlight and analyzde within 1 hour.

Cellular Binding and Uptake of gOBG3 as Detected by FluorescenceMicroscopy

Fluorecein isothiocyanate (FITC) conjugation of gOBG3: Purified gOBG3 at1 mg/mL concentration was labeled with FITC using Sigma's FluoroTag FITCconjugation kit (Stock No. FITC-1). Protocol outlined in the SigmaHandbook for small-scale conjugation was followed for gOBG3 labeling.

Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va.CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1 830)were seeded into 6 well plates at a cell density of 2×10⁵ cells perwell. C2C12 and Hepa-1-6 cells were cultured according to repository'sinstructions for 24-48 hours prior to analysis. Assay was performed whencells were 80% confluent.

FITC labeled gOBG3 cellular binding and uptake using microscopy: C2C12and Hepa 1-6 cells were incubated in the presence/absence of antibodydirected against human LSR (81B: N-terminal sequence of human LSR; doesnot cross react with mouse LSR and 93A: c-terminal sequence, crossreacts with mouse LSR) or an antiserum directed against gC1qr (953) for1 hour at 37° C., 5% CO₂. LSR antibodies were added to the media at aconcentration of 2 μg/mL. The anti-gC1qr antiserum was added to themedia at a volume of 2.5 μL undiluted serum (high concentration) or1:100 dilution (low concentration). Following incubation with specifiedantibody, FITC-gOBG3 (50 nM/mL) was added to each cell culture well.Cells were again incubated for 1 hour at 37° C., 5% C02. Cells werewashed 2× with PBS, cells were scraped from well into 1 mL of PBS. Cellsuspension was transferred to an eppendorf tube and centrifuged at 1000rpm for 2 minutes. Supernatant was removed and cells resuspended in 200μL of PBS. Binding and uptake of FITC-gOBG3 was analyzed by fluorescencemicroscopy under 40× magnification.

Analysis of C2C12 and Hepa 1-6 cells reveals identical phenotypes withrespect to FITC-gOBG3 binding and uptake profiles both in the presenceor absence of LSR antibodies. FITC-gOBG3 appears to be localized withinvesicles in the cytoplasm of both mouse hepatocytes and mouse myoblasts,suggesting that binding and uptake of FTTC-gOBG3 is occurring.FITC-gOBG3 uptake appears to be blocked when cells were pre-treated withthe anti-LSR antibody that recognizes mouse LSR. However, binding ofFTTC-gOBG3 to the cell surface does occur in a small portion of thecells (C2C12 and Hepa 1-6). At low concentration of the gC1qr antiserum,FITC-gOBG3 appears to be localized within vesicles in the cytoplasm ofboth cell types, similarly to the phenotype of cells that have notreceived antibody pre-treatment prior to addition of FITC-gOBG3.FITC-gOBG3 uptake and binding phenotype is not affected by pre-treatmentwith an LSR antibody that does not recognize mouse LSR. Together, thesedata suggest that uptake of FITC-gOBG3 can be blocked by a human LSRantibody which cross-reacts with mouse LSR. However, this phenotype isnot reproduced with other non cross-reactive LSR antibodies. Thus, thisassay may be useful for identifying agents that facilitate or preventthe uptake and/or binding of gOBG3 polypeptide fragments to cells.

Effect on LSR as a Lipoprotein Receptor

The effect of gOBG3 on the lipoprotein binding, internalizing anddegrading activity of LSR can also be tested. Measurement of LSR aslipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry31(19):4628-36; hereby incorporated herein in its entirety including anydrawings, tables, or figures). The effect of gOBG3 on the lipoproteinbinding, internalizing and degrading activity of LSR (or otherreceptors) can be compared with that of intact OBG3, with untreatedcells as an additional control. This assay can also be used to screenfor active and inhibitory variants of gOBG3, as well as agonists andantagonists of obesity-related activity.

Human liver PLC cells (ATCC Repository) were plated at a density of300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose)containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992).Media was changed on day 2. On day 3, the confluent monolayers werewashed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).Cells were incubated at 37° C. for 30 min with 10 ng/mL humanrecombinant leptin in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mMCaCl₂, 3.7 g/L sodium bicarbonate, pH 7.5, followed by another 30 rainincubation at 37° C. with increasing concentrations of gOBG3.Incubations were continued for 2 h at 37° C. after addition of 0.8 mMoleate and 20 μg/mL ¹²⁵-LDL. Monolayers were washed 2 timesconsecutively with PBS containing 0.2% BSA, followed by 1 wash withPBS/BSA, and then 2 times consecutively with PBS. The amounts ofoleate-induced binding, uptake and degradation of ¹²⁵I-LDL were measuredas previously described (Bihain & Yen, 1992, supra). Results are shownas the mean of triplicate determinations.

As shown in FIG. 6, the addition of gOBG3 leads to an increased activityof LSR as a lipoprotein receptor. The oleate-induced binding and uptakeof LDL appears more affected by gOBG3 as compared to the degradation.This increased LSR activity would potentially result in an enhancedclearance of triglyceride-rich lipoproteins during the postprandialstate. Thus, more dietary fat would be removed through the liver, ratherthan being deposited in the adipose tissue.

This assay could be used to determine the efficiency of a compound (oragonists or antagonists) to increase or decrease LSR activity (orlipoprotein uptake, binding and degradation through other receptors),and thus affect the rate of clearance of triglyceride-rich lipoproteins.

Effect on Muscle Differentiation

C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville,Md.) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine,pen/strep, etc)+10% FCS. Two days later they become 80-90% confluent. Atthis time, the media is changed to DMEM+2% horse serum to allowdifferentiation. The media is changed daily. Abundant myotube formationoccurs after 3-4 days of being in 2% horse serum, although the exacttime course of C2C12 differentiation depends on how long they have beenpassaged and how they have been maintained, among other things.

To test the effect of the presence of gACRP30 on muscle differentiation,gACRP30 (1 to 2.5 μg/mL) was added the day after seeding when the cellswere still in DMEM w/10% FCS. Two days after plating the cells (one dayafter gACRP30 was first added), at about 80-90% confluency, the mediawas changed to DMEM+2% horse serum plus gACRP30. The results show thatthe addition of gACRP30 causes the cells to begin organizing within oneday after its addition. In contrast to the random orientation of thecells not treated with gACRP30, those treated with gACRP30 alignedthemselves in relation to each other. In addition, differentiationoccurred after only 2 days of gACRP30 treatment, in contrast to the 3 to4 days needed in its absence.

Effect on Muscle Cell Fatty Acid Oxidation

C2C12 cells were differentiated in the presence or absence of 2 μg/mLgACRP30 for 4 days. On day 4, oleate oxidation rates were determined bymeasuring conversion of 1-¹⁴C-oleate (0.2 mM) to ¹⁴CO₂ for 90 min. C2C12cells differentiated in the presence of gACRP30 undergo 40% more oleateoxidation than controls differentiated in the absence of gACRP30. Thisexperiment can be used to screen for active fragments and peptides aswell as agonists and antagonists or activators and inhibitors of gOBG3polypeptides.

The effect of gACRP30 on the rate of oleate oxidation was compared indifferentiated C2C12 cells (murine skeletal muscle cells; ATCC,Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa 1-6; ATCC,Manassas, Va. CRL-1830). Cultured cells were maintained according tomanufacturer's instructions. The oleate oxidation assay was performed aspreviously described (Muoio et al (1999) Biochem J 338;783-791).Briefly, nearly confluent myocytes were kept in low serumdifferentiation media (DMEM, 2.5% Horse serum) for 4 days, at which timeformation of myotubes became maximal. Hepatocytes were kept in the sameDMEM medium supplemented with 10% FCS for 2 days. One hour prior to theexperiment the media was removed and 1 mL of preincubation media (MEM,2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA freeBSA, 0.25 mM Oleate, 5 μg/mL gentamycin) was added. At the start of theoxidation experiment ¹⁴C-Oleic acid (1 μCi/mL, American RadiolabeledChemical Inc., St. Louis, Mo.) was added and cells were incubated for 90min at 37° C. in the absence/presence of 2.5 μg/mL gACRP30. After theincubation period 0.75 mL of the media was removed and assayed for¹⁴C-oxidation products as described below for the muscle FFA oxidationexperiment. Oleate oxidation in C2C12 cells determined over 90 minincreased significantly (39%; p=0.036, two-tailed t-Test) in cellstreated with gACRP30. In contrast, no detectable increase in the rate ofFFA oxidation was seen in hepatocytes incubated with gACRP30.

Triglyceride and Protein Analysis Following Oleate Oxidation in CulturedCells

Following transfer of media for oleate oxidation assay, cells wereplaced on ice. To determine triglyceride and protein content, cells werewashed with 1 mL of 1×PBS to remove residual media. To each well 300 μLof cell dissociation solution (Sigma) was added and incubated at 37° C.for 10 min. Plates were tapped to loosen cells, and 0.5 mL of 1×PBS wasadded. The cell suspension was transferred to an eppendorf tube, eachwell was rinsed with an additional 0.5 mL of 1×PBS, and was transferredto appropriate eppendorf tube. Samples were centrifuged at 1000 rpm for10 minutes at room temperature. Supernatant was discarded and 750 μL of1×PBS/2% chaps was added to cell pellet. Cell suspension was vortexedand place on ice for 1 hour. Samples were then centrifuged at 13000 rpmfor 20 min at 4° C. Supernatants were transferred to new tube and frozenat −20° C. until analyzed. Quantitative measure of triglyceride level ineach sample was determined using Sigma Diagnostics GPO-TRINDER enzymatickit. The procedure outlined in the manual was adhered to, with thefollowing exceptions: assay was performed in 48 well plate, 350 μL ofsample volume was assayed, control blank consisted of 350 μL PBS/2%chaps, and standard contained 10 μL standard provide in kit plus 690 μLPBS/2% chaps. Analysis of samples was carried out on a Packard SpectraCount at a wavelength of 550 nm. Protein analysis was carried out on 25μL of each supernatant sample using the BCA protein assay (Pierce)following manufacturer's instructions. Analysis of samples was carriedout on a Packard Spectra Count at a wavelength of 550 nm.

Triglyceride production in both C2C12 and Hepa 1-6 cells did not changesignificantly in the absence/presence of ACRP30 and gACRP30. The proteincontent of all cells analyzed was equivalent in the absence/presence ofACRP30 and gACRP30.

In Vitro Glucose Uptake by Muscle Cells

L6 Muscle cells are obtained from the European Culture Collection(Porton Down) and are used at passages 7-11. Cells are maintained instandard tissue culture medium DMEM, and glucose uptake is assessedusing [³H-2-deoxyglucose (2DG) with or without gOBG3 polypeptidefragment in the presence or absence of insulin (10⁻⁸ M) as has beenpreviously described (Walker, P. S. et al. (1990) Glucose transportactivity in L6 muscle cells is regulated by the coordinate control ofsubcellular glucose transporter distribution, biosynthesis, and mRNAtranscription. JBC 265(3):1516-1523; and Kilp, A. et al. (1992)Stimulation of hexose transport by metformin in L6 muscle cells inculture. Endocrinology 130(5):2535-2544, which disclosures are herebyincorporated by reference in their entireties). Uptake of 2DG isexpressed as the percentage change compared with control (no addedinsulin or gOBG3 polypeptide fragment). Values are presented as mean±SEMof sets of 4 wells per experiment. Differences between sets of wells areevaluated by Student's t test, probability values p<0.05 are consideredto be significant.

Example 5 Effect of gOBG3 on Mice Fed a High-Fat Diet

Experiments are performed using approximately 6 week old C57B1/6 mice (8per group). All mice are housed individually. The mice are maintained ona high fat diet throughout each experiment. The high fat diet (cafeteriadiet; D12331 from Research Diets, Inc.) has the following composition:protein kcal % 16, sucrose kcal % 26, and fat kcal % 58. The fat wasprimarily composed of coconut oil, hydrogenated.

After the mice are fed a high fat diet for 6 days, micro-osmotic pumpsare inserted using isoflurane anesthesia, and are used to provide gOBG3,OBG3, saline, and an irrelevant peptide to the mice subcutaneously(s.c.) for 18 days. gOBG3 is provided at doses of 50, 25, and 2.5μg/day; OBG3 is provided at 100, 50, and 5 μg/day; and the irrelevantpeptide is provided at 10 μg/day. Body weight is measured on the first,third and fifth day of the high fat diet, and then daily after the startof treatment. Final blood samples are taken by cardiac puncture and areused to determine triglyceride (TG), total cholesterol (TC), glucose,leptin, and insulin levels. The amount of food consumed per day is alsodetermined for each group.

In a preliminary experiment, mice treated with 2.5 μg/day gOBG3 hadsignificantly lowered body weight.

Example 6 Tests of Obesity-Related Activity in Humans

Tests of the efficacy of gOBG3 in humans are performed in accordancewith a physician's recommendations and with established guidelines. Theparameters tested in mice are also tested in humans (e.g. food intake,weight, TG, TC, glucose, insulin, leptin, FFA). It is expected that thephysiological factors would show changes over the short term. Changes inweight gain might require a longer period of time. In addition, the dietwould need to be carefully monitored. Globular OBG3 would be given indaily doses of about 6 mg protein per 70 kg person or about 10 mg perday. Other doses would also be tested, for instance 1 mg or 5 mg per dayup to 20 mg, 50 mg, or 100 mg per day.

Example 7 In Vivo Tests for Obesity-Related Activity in Rodent DiabetesModels

As metabolic profiles differ among various animal models of obesity anddiabetes, analysis of multiple models is undertaken to separate theeffects of gOBG3 polypeptide fragment on hyperglycemia,hyperinsulinemia, hyperlipidemia, and obesity. Mutation in colonies oflaboratory animals and different sensitivities to dietary regimens havemade the development of animal models with non-insulin dependentdiabetes associated with obesity and insulin resistance possible.Genetic models such as db/db and ob/ob (See Diabetes (1982) 31(1):1-6)in mice and fa/fa in zucker rats have been developed by the variouslaboratories for understanding the pathophysiology of disease and fortesting the efficacy of new antidiabetic compounds (Diabetes (1983) 32:830-838; Annu Rep Sankyo Res Lab (1994) 46:1-57). Homozygous C57BL/KsJ-db/db mice developed by Jackson Laboratory are obese,hyperglycemic, hyperinsulinemric, and insulin resistant (J Clin Invest(1990) 85:962-967), whereas heterozygous mice are lean andnormoglycemic. In db/db model, mice progressively develop insulinopeniawith age, a feature commonly observed in late stages of human type IIdiabetes when blood sugar levels are insufficiently controlled. Thestate of the pancreas and its course vary according to the models. Sincethis model resembles that of type II diabetes mellitus, the compounds ofthe present invention are tested for blood sugar and triglycerideslowering activities. Zucker (fa/fa) rats are severely obese,hyperinsulinemic, and insulin resistant (Coleman, Diabetes 31:1, 1982;E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds.(Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990),pp.299-340), and the fa/fa mutation may be the rat equivalent of themurine db mutation (Friedman et al. Cell 69:217-220, 1992; Truett etal., Proc Natl Acad Sci USA 88:7806, 1991). Tubby (tub/tub) mice arecharacterized by obesity, moderate insulin resistance andhyperinsulinemia without significant hyperglycemia (Coleman et al., JHeredity 81:424, 1990).

Previously, leptin was reported to reverse insulin resistance anddiabetes mellitus in mice with congenital lipodystrophy (Shimomura etal. Nature 401:73-76 (1999); hereby incorporated herein in its entiretyincluding any drawings, figures, or tables). Leptin was found to be lesseffective in a different lipodystrophic mouse mode] of lipoatrophicdiabetes (Gavrilova et al Nature 403:850 (2000); hereby incorporatedherein in its entirety including any drawings, figures, or tables). Theinstant invention encompasses the use of gOBG3 polypeptide fragments forreducing the insulin resistance and hyperglycaemia in this model eitheralone or in combination with leptin, the leptin peptide (U.S.Provisional Application No 60/155,506), or other compounds. Assaysinclude that described previously in Gavrilova et al. ((2000) Diabetes49(11):1910-6; (2000) Nature 403(6772):850) using A-ZIP/F-1 mice, exceptthat gOBG3 polypeptide fragment would be administered using the methodspreviously described in Example 5 (or Examples 8-10). The glucose andinsulin levels of the mice would be tested, and the food intake andliver weight monitored, as well as other factors, such as leptin, FFA,and TG levels, typically measured in our experiments (see Example 5,above, or Examples 8-10).

The streptozotocin (STZ) model for chemically-induced diabetes is testedto examine the effects of hyperglycemia in the absence of obesity.STZ-treated animals are deficient in insulin and severely hyperglycemic(Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H.Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc.,New York, ed. 4, 1990), pp. 299-340). The monosodium glutamate (MSG)model for chemically induced obesity (Olney, Science 164:719, 1969,Cameron et al., Clin Exp Pharmacol Physiol 5:41, 1978), in which obesityis less severe than in the genetic models and develops withouthyperphagia, hyperinsulinemia and insulin resistance, is also examined.Finally, a non-chemical, non-genetic model for induction of obesityincludes feeding rodents a high fa/high carbohydrate (cafeteria diet)diet ad libitum.

The instant invention encompasses the use of gOBG3 polypeptide fragmentfor reducing the insulin resistance and hyperglycemia in any or all ofthe above rodent diabetes models or in humans with Type I or Type IIdiabetes or other preferred metabolic diseases described previously ormodels based on other mammals. In the compositions of the presentinvention the gOBG3 polypeptide fragment may, if desired, be associatedwith other compatible pharmacologically-active antidiabetic agents suchas insulin, leptin (U.S. Provisional Application No. 60/155,506), ortroglitazone, either alone or in combination. Assays include thatdescribed previously in Gavrilova et al. ((2000) Diabetes 49(11):1910-6;(2000) Nature 403(6772):850) using A-ZIP/F-1 mice, except that gOBG3polypeptide fragment is administered intraperitoneally (i.p.),subcutaneously (s.c.), intramuscularly (i.m.), or intravenously (i.v.).The glucose and insulin levels of the mice would be tested, and the foodintake and liver weight monitored, as well as other factors, such asleptin, FFA, and TG levels, typically measured in our experiments.

In Vivo Assay for Anti-Hyperglycemic Activity of gOBG3 PolypeptideFragment

Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old)are housed (7-9 mice/cage) under standard laboratory conditions at 22°C. and 50% relative humidity, and maintained on a diet of Purina rodentchow and water ad libitum. Prior to treatment, blood is collected fromthe tail vein of each animal and blood glucose concentrations aredetermined using One Touch Basic Glucose Monitor System (Lifescan). Micethat have plasma glucose levels between 250 to 500 mg/dl are used. Eachtreatment group consists of seven mice that are distributed so that themean glucose levels are equivalent in each group at the start of thestudy. db/db mice are dosed by micro-osmotic pumps, inserted usingisoflurane anesthesia; to provide gOBG3 polypeptide fragment, saline,and an irrelevant peptide to the mice subcutaneously (s.c.). Blood issampled from the tail vein hourly for 4 hours and at 24, 30 hpost-dosing and analyzed for blood glucose concentrations. Food iswithdrawn from 0-4 h post dosing and reintroduced thereafter. Individualbody weights and mean food consumption (each cage) are also measuredafter 24 h. Significant differences between groups (comparing gOBG3fragment treated to saline-treated) are evaluated using Student t-test.

In Vivo Insulin Sensitivity Assay

In vivo insulin sensitivity is examined by utilizing two-stephyperinsulinemic-euglycemic clamps according to the following protocol.Rodents from any or all of the various models described in Example 2 arehoused for at least a week prior to experimental procedures. Surgeriesfor the placement of jugular vein and carotid artery catheters areperformed under sterile conditions using ketamine and xylazine (i.m.)anesthesia. After surgery, all rodents are allowed to regainconsciousness and placed in individual cages. gOBG3 polypeptide fragmentor vehicle is administered through the jugular vein after completerecovery and for the following two days. Sixteen hours after the lasttreatment, hyperinsulinemic-euglycemic clamps are performed. Rodents areplaced in restrainers and a bolus of 4 μCi [3-³H] glucose (NEN) isadministered, followed by a continuous infusion of the tracer at a doseof 0.2 μCi/min (20 μl/min). Two hours after the start of the tracerinfusion, 3 blood samples (0.3 ml each) are collected at 10 minuteintervals (−20-0 min) for basal measurements. An insulin infusion isthen started (5 mU/kg/min), and 100 μl blood samples are taken every 10min. to monitor plasma glucose. A 30% glucose solution is infused usinga second pump based on the plasma glucose levels in order to reach andmaintain euglycemia. Once a steady state is established at 5 mU/kg/mininsulin (stable glucose infusion rate and plasma glucose), 3 additionalblood samples (0.3 ml each) are obtained for measurements of glucose,[3-³H] glucose and insulin (100-120 min.). A higher dose of insulin (25mU/kg/min.) is then administered and glucose infusion rates are adjustedfor the second euglycemic clamp and blood samples are taken at 220-240min. Glucose specific activity is determined in deproteinized plasma andthe calculations of Rd and hepatic glucose output (HGO) are made, asdescribed (Lang et al., Endocrinology 130:43, 1992). Plasma insulinlevels at basal period and after 5 and 25 mU/kg/min. infusions are thendetermined and compared between gOBG3 fragment treated and vehicletreated rodents.

Insulin regulation of glucose homeostasis has two major components;stimulation of peripheral glucose uptake and suppression of hepaticglucose output. Using tracer studies in the glucose clamps, it ispossible to determine which portion of the insulin response is affectedby gOBG3 polypeptide fragment.

Example 8 Effect of gOBG-3 on Plasma Free Fatty Acid in C57 BL/6 Mice

The effect of the globular head of ACRP30 on postprandial lipemia (PPL)in normal C57BL6/J mice was tested. ACRP30 is another name for adipo Qand is the mouse protein homologue to the human APM1 protein. OBG3 is ageneric way to refer to all of these forms. The globular head form isindicated by placing a ‘g’ in front, e.g. gACRP30 or gOBG3. The gOBG3used was prepared by proteolytic digestion of recombinant OBG3 asdescribed previously in Example 2. Acetylated trypsin was used asprotease.

The mice used in this experiment were fasted for 2 hours prior to theexperiment after which a baseline blood sample was taken. All bloodsamples were taken from the tail using EDTA coated capillary tubes (50μL each time point). At time 0 (8:30 AM), a standard high fat meal (6 gbutter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 mLdistilled water prepared fresh following Nb#6, JF, pg. 1) was given bygavage (vol.=1% of body weight) to all animals.

Immediately following the high fat meal, 25 μg gOBG3 was injected i.p.in 100 μL saline. The same dose (25 μg/mL in 100 μL) was again injectedat 45 min and at 1 hr 45 min (treated group, n=8). Control animals (n=8)were injected with saline (3×100 μL). Untreated and treated animals werehandled in an alternating mode.

Blood samples were taken in hourly intervals, and were immediately puton ice. Plasma was prepared by centrifugation following each time point.Plasma was kept at −20° C. and free fatty acids (FFA), triglycerides(TG) and glucose were determined within 24 hours using standard testkits (Sigma and Wako). Due to the limited amount of plasma available,glucose was determined in duplicate using pooled samples. For each timepoint, equal volumes of plasma from all 8 animals per treatment groupwere pooled. Error bars shown for glucose therefore represent the SD ofthe duplicate determination and not the variation between animals as forTG and FFA.

Results

The increase in plasma FFA due to the high fat meal was significantlylower in mice treated with gOBG3 at all time points between 1 and 4 hr.This can be interpreted as increase in FFA oxidation (FIG. 8).

Treatment with gOBG3 also led to a significantly smaller increase inplasma TG compared to untreated mice. However, this effect was lesspronounced than the effect on FFA (FIG. 9).

Glucose turnover was significantly improved following treatment withgOBG3; this effect can be interpreted as improved insulin sensitivitypossibly due to the decrease in FFA FIG. 10).

Similar results were seen previously in a prior experiment involvingonly 2 treatments (at 0 and at 45 minutes; data not shown). A strong FFAlowering effect of gOBG3 coupled with a less dominant TG lowering effectwas observed.

Example 9 Effect of gOBG3 on Plasma Leptin and Insulin in C57 BL/6 Mice

The effect of the globular head of ACRP30 on plasma leptin and insulinlevels during postprandial lipemia (PPL) in normal C57BL6/J mice wastested. The experimental procedure was the same as that described inExample 8, except that blood was drawn only at 0, 2 and 4 hours to allowfor greater blood samples needed for the determination of leptin andinsulin by RIA.

Briefly, 16 mice were fasted for 2 hours prior to the experiment afterwhich a baseline blood sample was taken. All blood samples were takenfrom the tail using EDTA coated capillary tubes (100 μL each timepoint). At time 0 (9:00 AM), a standard high fat meal (see Example 8)was given by gavage (vol.=1% of body weight) to all animals.Inmmediately following the high fat meal, 25 μg gOBG3 was injected i.p.in 100 μL saline. The same dose (25 μg in 100 μL) was again injected at45 min and at I hr 45 min (treated group, n=8). Control animals (n=8)were injected with saline (3×100 μL). Untreated and treated animals werehandled in an alternating mode.

Blood samples were immediately put on ice and plasma was prepared bycentrifugation following each time point. Plasma was kept at −20° C. andfree fatty acids (FFA) were determined within 24 hours using a standardtest kit (Wako). Leptin and Insulin were determined by RIA (ML-82K andSRI-13K, LINCO Research, Inc., St. Charles, Mo.) following themanufacturer's protocol. However, only 20 μL plasma was used. Eachdetermination was done in duplicate. Due to the limited amount of plasmaavailable, leptin and insulin were determined in 4 pools of 2 animalseach in both treatment groups.

Results

As shown previously (Example 8), treatment with gOBG3 significantlyreduced the postprandial increase in plasma FFA caused by the high fatmeal at 2 hours (FIG. 11). There was no significant change in plasmaleptin levels at any time point; treatment with gOBG3 did not affectleptin levels (FIG. 12). Insulin levels (FIG. 13) indicate a marginalincrease in insulin at 2 hours. However, when analyzed as percentagechange from t₀, this increase (212% vs. 260%, control vs. treated) wasstatistically not significant (p=0.09). These data reconfirm thepreviously shown acceleration of FFA metabolism by treatment with gOBG3.They also show that gOBG3 does not affect leptin and insulin plasmalevels and that gOBG3 reduces hyperglycemia during postprandial lipemiaand also induces weight loss during treatment over several days. Withoutbeing limited by any particular theory, the data suggests: a) that thereduction in weight is caused by a leptin independent increase inmetabolism; and b) that gOBG3 leads to increased insulin sensitivity.

Example 10 Effect of OBG3 on Plasma FFA, TG and Glucose in C57 BL/6 Mice

The effect of the globular head of ACRP30 on plasma FFA, TG, glucose,leptin and insulin levels during postprandial lipemia (PPL) in normalC57BL6/J mice has been described. Weight loss resulting from gOBG3 (2.5μg/day) given to normal C57BL6/J mice on a high fat diet has also beenshown (Example 5). In comparison, a much higher dose of the completeform of ACRP30 (200 μg/day) was needed to induce a relatively smallereffect in mice. This example shows the effect of the ACRP30-completeform on plasma FFA, TG and glucose levels.

The experimental procedure was similar to that described in Example 8.Briefly, 14 mice were fasted for 2 hours prior to the experiment afterwhich a baseline blood sample was taken. All blood samples were takenfrom the tail using EDTA coated capillary tubes (50 μL each time point).At time 0 (9:00 AM), a standard high fat meal (see Example 8) was givenby gavage (vol.=1% of body weight) to all animals. Immediately followingthe high fat meal, 4 mice were injected 25 μg OBG3 i.p. in 100 μLsaline. The same dose (25 μg in 100 μL) was again injected at 45 min andat 1 hr 45 min. A second treatment group (n=4) received 3 times 50 μgOBG3 at the same intervals. Control animals (n=6) were injected withsaline (3×100 μL). Untreated and treated animals were handled in analternating mode.

Blood samples were immediately put on ice. Plasma was prepared bycentrifugation following each time point. Plasma was kept at −20° C. andfree fatty acids (FFA), triglycerides (TG) and glucose were determinedwithin 24 hours using standard test kits (Sigma and Wako).

Results

Treatment with full-length OBG3 had no effect on plasma FFA levels (FIG.14) except for t=2 hours when a statistically significant reduction wasshown (p<0.05). No significant change in postprandial TG (FIG. 15) andglucose levels (FIG. 16) was seen in treated animals. The data presentedshow that the complete form of OBG3 did not reduce FFA, TG and glucoselevels in contrast to what was observed for the globular domain(Examples 5, 8, 9). Only at 2 hours post-gavage, did treatment with OBG3reduce FFA plasma concentrations significantly (p<0.05). These resultsdemonstrate that gOBG3 is much more active in vivo than the full-lengthprotein. A similar effect was seen for body weight reduction; theglobular head was much more active than the full-length protein.

Example 11 Effect of gACRP30 on FFA following Epinephrine Injection

In mice, plasma free fatty acids increase after intragastricadministration of a high fat/sucrose test meal. These free fatty acidsare mostly produced by the activity of lipolytic enzymes i.e.lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, theseenzymes are found in significant amounts both bound to endothelium andfreely circulating in plasma. Another source of plasma free fatty acidsis hormone sensitive lipase (HSL) that releases free fatty acids fromadipose tissue after β-adrenergic stimulation. To test whether gACRP30also regulates the metabolism of free fatty acid released by HSL, micewere injected with epinephrine.

Two groups of mice (n=5 each) were given epinephrine (5 μg) byintraperitoneal injection. A treated group was injected with gACRP30 (25μg) one hour before and again together with epinephrine, while controlanimals received saline. Plasma was isolated and free fatty acids andglucose were measured as described above (Example 10). As shown in FIG.18, epinephrine injections (5 μg) caused an increase in plasma freefatty acids and glucose. Both effects were significantly reduced ingACRP30-treated mice.

This reduction in the increases of glucose and FFA levels was not due toblockage of the β-adrenergic effect of epinephrine, as shown by inducingthe release of FFA from isolated adipose tissue in vitro. In thesecontrol studies, adipose tissue was removed from normal C57BL/6J miceand incubated in Krebs-Henseleit bicarbonate buffer. Epinephrine wasadded and the concentration of FFA in the medium following a 90 minincubation was determined. Epinephrine (10 uM) caused a 1.7-foldincrease in free fatty acids in the media. Increasing concentrations ofgACRP30 or ACRP30 up to 50 μg/ml did not inhibit this effect ofepinephrine.

The data presented thus far indicate that the globular domain of ACRP30exerts profound pharmacological effects on the metabolism of energysubstrates with the most evident effect on plasma free fatty acids.Further, the reduction in plasma FFA concentration cannot be explainedby inhibition of either LPL which would cause an increase in plasmatriglycerides while a decrease of plasma triglycerides is actuallyobserved, or by inhibition of HSL. Thus, the simplest explanation isthat gACRP30 causes increased removal of free fatty acids from thecirculation by promoting cellular uptake.

Example 12 Effect of gACRP30 on Muscle FFA Oxidation

To investigate the effect of gACRP30 on muscle free fatty acidoxidation, intact hind limb muscles from C57BL/6J mice were isolated andFFA oxidation was measured using oleate as substrate [Clee, et al.,“Plasma and vessel wall lipoprotein lipase have different roles inatherosclerosis”, J Lipid Res 41, 521-531 (2000); Muoio, et al., “Leptinopposes insulin's effects on fatty acid partitioning in muscles isolatedfrom obese ob/ob mice”, Am J Physiol 276, E913-921 (1999). Oleateoxidation in isolated muscle was measured as previously described[Cuendet, et al., “Decreased basal, noninsulin-stimulated glucose uptakeand metabolism by skeletal soleus muscle isolated fromobese-hyperglycemic (ob/ob) mice”, J Clin Invest 58, 1078-1088 (1976);Le Marchand-Brustel, et al., “Insulin binding and effects in isolatedsoleus muscle of lean and obese mice”, Am J Physiol 234, E348-E358(1978)). Briefly, mice were sacrificed by cervical dislocation andsoleus and EDL muscles were rapidly isolated from the hind limbs. Thedistal tendon of each muscle was tied to a piece of suture to facilitatetransfer among different media. All incubations were carried out at 30°C. in 1.5 mL of Krebs-Henseleit bicarbonate buffer (118.6 mM NaCl, 4.76mM KCl, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 2.54 mM CaCl₂, 25 mM NaHCO₃, 10mM Hepes, pH 7.4) supplemented with 4% FFA free bovine serum albumin(fraction V, RIA grade, Sigma) and 5 mM glucose (Sigma). The totalconcentration of oleate (Sigma) throughout the experiment was 0.25 mM.All media were oxygenated (95% O₂; 5% CO₂) prior to incubation. The gasmixture was hydrated throughout the experiment by bubbling through a gaswasher (Kontes Inc., Vineland, N.J.).

Muscles were rinsed for 30 min. in incubation media with oxygenation.The muscles were then transferred to fresh media (1.5 mL) and incubatedat 30° C. in the presence of 1 μCi/mL [1-¹⁴C] oleic acid (AmericanRadiolabeled Chemicals). The incubation vials containing this media weresealed with a rubber septum from which a center well carrying a piece ofWhatman paper (1.5 cm×11.5 cm) was suspended.

After an initial incubation period of 10 min with constant oxygenation,gas circulation was removed to close the system to the outsideenvironment and the muscles were incubated for 90 min at 30° C. At theend of this period, 0.45 mL of Solvable (Packard Instruments, Meriden,Conn.) was injected onto the Whatman paper in the center well and oleateoxidation by the muscle was stopped by transferring the vial onto ice.

After 5 min, the muscle was removed from the medium, and an aliquot of0.5 mL medium was also removed. The vials were closed again and 1 mL of35% perchloric acid was injected with a syringe into the media bypiercing through the rubber septum. The CO₂ released from the acidifiedmedia was collected by the Solvable in the center well. After a 90 mincollection period at 30° C., the Whatman paper was removed from thecenter well and placed in scintillation vials containing 15 mL ofscintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.).The amount of ¹⁴C radioactivity was quantitated by liquid scintillationcounting. The rate of oleate oxidation was expressed as nmol oleateproduced in 90 min/g muscle.

To test the effect of gACRP30 or ACRP30 on oleate oxidation, theseproteins were added to the media at a final concentration of 2.5 μg/mLand maintained in the media throughout the procedure.

Two muscles of different oxidative capacity (soleus and extensordigitorum longus (EDL)) were tested (FIG. 19). EDL and Soleus muscleswere isolated from both legs of normal C57BL/6J mice (n=18). One muscleof each pair was incubated in medium with 2.5 μg/mL gACRP30 (dark gray)and one in medium without gACRP30 (control—light gray). Thisexperimental design allowed us to compare oleate oxidation in pairs ofmuscles isolated from the same animal. ¹⁴C-Oleate oxidation wasdetermined over 90 minutes. Incubation of EDL and soleus muscles for 90minutes in medium containing 2.5 μg/ml gACRP30 leads to a statisticallysignificant increase in oleate oxidation (p<0.05, paired, one-tailed,t-Test) or (p=0.0041, Repeated Measures Analysis of Variance, UnivariateTests of Hypotheses for Within Subject Effects) in both muscle types.

Both muscle types showed a significant response to gACRP30. The relativeincrease in FFA oxidation was 17% (p=0.03) and 10% (p=0.04) for EDL andsoleus, respectively. In humans, muscles represent approximately 25% ofbody weight. Therefore, even a moderate increase in free fatty acidoxidation can have quantitatively important consequences on overallenergy utilization.

Example 13 Effect of gArcp30 on Triglyceride in Muscle and LiverIsolated from Mice

To determine whether the increased FFA oxidation induced by gACRP30 isalso accompanied by increased FFA delivery into muscle or liver, thehindlimb muscle and liver triglyceride content was measured aftergACRP30 treatment of mice. Hind limb muscles as well as liver sampleswere removed from treated and untreated animals and the triglyceride andfree fatty acid concentration was determined following a standard lipidextraction method Shimabukuro, et al., “Direct antidiabetic effect ofleptin through triglyceride depletion of tissues”, Proc Natl Acad SciUSA 94, 4637-4641 (1997) followed by TG and FFA analysis using standardtest kits.

Short-term treatment of animals with gACRP30 (2 injections of 25 μg eachgiven within 3 hours before sacrifice) did not change the triglyceridecontent either of hind limb muscle or liver tissue (data not shown).However, after 3 days of treatment, during which period normal C57BL/6Jmice consumed a regular rodent diet, mice that had received 25 μg ofgACRP30 twice daily showed significantly higher (p=0.002) muscletriglyceride content, (FIG. 20A) than those receiving saline (control:light gray; gACRP30: dark gray). This contrasted with a lack of increasein liver triglycerides (FIG. 20B). Furthermore, no detectable increasein muscle TG was observed after the 16-day treatment shown independentlyby directly measuring the muscle TG content and by oil red O staining offrozen microscope sections. In summary, the data indicate that theincrease in TG content was transient.

These data are consistent with the notion that gACRP30 increases therate of removal of free fatty acids from plasma at least partly byincreasing their delivery to the muscle; much of the FFAs areimmediately oxidized while some are stored as triglycerides andsubsequently oxidized. Further support for this interpretation wasobtained by measuring the concentration of ketone bodies in plasma oftreated and untreated animals following a high fat/sucrose meal.

Ketone bodies (KB) are produced in the liver as a result of free fattyacid oxidation, but KB formation does not occur significantly in muscle.In mice receiving the high fat test meal and saline injection, the levelof plasma KB increased significantly over the next 3 hours (183±12%,n=6). Animals treated with gACRP30, on the other hand, showed noincrease in plasma KB concentrations. Thus, gACRP30 inhibits eitherdirectly KB formation or can decrease KB production by inhibiting liverFFA oxidation.

Example 14 Effect of gACRP30 on Weight Gain and Weight Loss of Mice andon Maintenance of Weight Loss in Mice

Two independent studies showed that gACRP30 also affects overall energyhomeostasis. In the first, 10-week-old male C57BL/6J mice were put on avery high fat/sucrose purified diet for 19 days to promote weight gain(see Example 5); the average body weight at this time was 30 g. The micewere then surgically implanted with an osmotic pump (Alzet, Newark,Del.) delivering either 2.5 μg/day of gACRP30, 5 μg/day of ACRP30, orphysiological saline. The mice were continued on the high fat diet andtheir body weight was recorded over the following 10-day period.

Mice treated with saline or 5 μg/day of full-length ACRP30 continued togain weight at an average daily rate of 0.16% and 0.22%, respectively.In contrast, mice treated with gACRP30 experienced a significant weightreduction (−3.7%, p=0.002) during the first 4 days and then their weightremained constant (FIG. 21A). Thus, in this inbred strain of normalmice, a continuous infusion of a daily low dose of gACRP30 can preventweight gain caused by high fat/sucrose feeding, in a sustainable way.

This result was confirmed and extended in a second study performed inmature 9 month old, male obese C57BL/6J mice that had been on the samehigh fat/sucrose diet for 6 months; the average body weight when thestudy began was 52.5±0.8 g. Three groups of 8 mice were treated withsaline, ACRP30 or gACRP30 for 16 days. Animals in the treated groupreceived twice daily 25 μg of protein subcutaneously. Body weights wererecorded at the indicated time points.

Treatment with gACRP30 led to significant (p<0.05) weight loss at day 3.This effect became even more significant as the study continued. Duringthe 16 day study period, the obese C57BL/6J mice that received gACRP30lost about 8% (p=0.001) of their initial body weight despite the factthat they were maintained on a high fat/sucrose diet (FIG. 21B). Salinetreated animals showed only marginal fluctuations in their body weight(p=n.s.). Animals treated with the full-length ACRP30, but at a 10-foldhigher dose than that used in the first experiment, also lostsignificant weight (−3.2%, p=0.025). Interestingly, mice treated withgACRP30 continued to lose weight at a steady rate during the 16-daystudy period, while the rate of weight reduction in those treated withthe full-length ACRP30 decreased during the later phase of the study.Food consumption in gACRP30 treated animals was not significantlydifferent from saline or ACRP30 treated animals (FIG. 21D).

Treatment with gACRP30 caused a significant reduction in theconcentration of plasma free fatty acids (FIG. 21C). This effect wassignificant after 3 days of treatment (p<0.05 vs. saline) and continuedthroughout the complete study period. Shown is the plasma FFA level atday 16 of the study. The initial FFA plasma concentration was the samein all three treatment groups. It should be noted, however, that despitethis reduction the plasma free fatty acid concentration of thesemassively obese animals remains about 40-60% higher than that of normalmice. A blood chemistry analysis (including determination of SOPT, SGOT,urea, creatinine or bilirubin) performed on the terminal blood samplesdid not reveal any abnormal plasma parameters (FIG. 22).

Data are expressed throughout as mean±SEM; a p-value<0.05 was consideredstatistically significant. Statistical analysis was typically done usingeither the unpaired Student's t test or the paired Student's t test, asindicated in each study.

Maintenance of Weight Loss in Mice

In order to demonstrate the weight loss maintaining properties ofgACRP30, normal mice are put on a reduced calorie diet to promote weightloss. The reduced calorie diet is continued until the mice lose 10% oftheir intitial weight. A second group of mice are continued on theweight reduced diet until the mice lose 20% of their intitial weight.The mice are then surgically implanted with an osmotic pump (Alzet,Newark, Del.) delivering either 2.5 μg/day of gACRP30, 5 μg/day ofACRP30, or physiological saline. The mice are returned to a normal dietand their body weights are recorded over a 10-day period. After 10 days,the result that mice that have been treated with gACRP30 have a lowerweight than mice that were treated with saline will be taken to provideevidence that treatment with gACRP30 promotes the maintenance of weightloss.

Example 15 Effect of gACRP30 on FFA following Intralipid Injection

Two groups of mice (n=5 each) were intravenously (tail vein) injectedwith 30 μL bolus of Intralipid-20% (Clintec) to generate a sudden risein plasma FFAs, thus by-passing intestinal absorption. Intralipid is anintravenous fat emulsion used in nutritional therapy). A treated group(♦ gACRP30-treated) was injected with gACRP30 (25 μg) at 30 and 60minutes before Intralipid was given, while control animals (▴ control)received saline. Plasma was isolated and FFAs were measured as describedpreviously.

The effect of gACRP30 on the decay in plasma FFAs following the peakinduced by Intralipid injection was then monitored. As shown in FIG. 23,gACRP30 accelerates the removal of FFAs from plasma after Intralipidinjection. Thus, gACRP30 accelerates the clearance of FFAs withoutinterfering with intestinal absorption. Although not wishing to be boundby any theory, because Intralipid does not elicit a significant insulinresponse, the results also indicate that gACRP30 regulation of FFAmetabolism occurs independently of insulin.

Example 16 Solubilization of OBG3 and Fragments Thereof

Vector construction: Polynucleotides encoding polypeptides selected fromamino acids 18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244,25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244,34-244, 35-244, 36-244, 37-244, 38-244, 39-40-244, 41-244, or 42-244 ofSEQ ID NO:2 (APM1) wherein the cysteine at position 36 is substituted byan amino acid other than cysteine; amino acids 37-244, 38-244, 39-244,40-244, 41-244, or 42-244 of SEQ ID NO:2; amino acids 18-247, 19-247,20-247, 21-247, 22-247, 23-247, 24-247, 25-247, 26-247, 27-247, 28-247,29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247, 36-247, 37-247,38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQID NO:4 (ACRP30) wherein the cysteine at position 39 is substituted byan amino acid other than cysteine; or amino acids 40-247, 41-247,42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4 are cloned intobacterial expression vector pTrcHis.

Polynucleotides encoding polypeptides selected from amino acids 18-244,19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244, 27-244,28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244, 36-244,37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2 (APM1)wherein the cysteine at position 36 is substituted by an amino acidother than cysteine; amino acids 37-244, 38-244, 39-244, 40-244, 41-244,or 42-244 of SEQ ID NO:2; amino acids 18-247, 19-247, 20-247, 21-247,22-247, 23-247, 24-247, 25-247, 26-247, 27-247, 28-247, 29-247, 30-247,31-247, 32-247, 33-247, 34-247, 35-247, 36-247, 37-247, 38-247, 39-247,40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4(ACRP30) wherein the cysteine at position 39 is substituted by an aminoacid other than cysteine; or amino acids 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 are cloned into a modified bacterialexpression vector pET30a (providing a His Tag at the C-terminus of theAPM1 or ACRP30 polypeptide).

Polynucleotides encoding polypeptides selected from amino acids 1-244 ofSEQ ID NO:2 (APM1) or amino acids 1-247 of SEQ ID NO:4 (ACRP30) arecloned into Baculoviral expression vector FastBacHT. Polynucleotidesencoding a heterologous polypeptide comprised of human zinc-alpha2-glycoprotein signal peptide fused N-terminally to gOBG3 polypeptidefragment of the invention, wherein said gOBG3 polypeptide fragment isselected from amino acids 18-244, 19-244, 20-244, 21-244, 22-244,23-244, 24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244,32-244, 33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244,41-244, or 42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position36 is substituted by an amino acid other than cysteine; amino acids37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2; aminoacids 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247,35-247, 36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine atposition 39 is substituted by an amino acid other than cysteine; oramino acids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ IDNO:4, are cloned into Baculoviral expression vector FastBacHT.

Polynucleotides encoding polypeptides comprising amino acids 1-244 ofSEQ ID NO:2 (APM1) or amino acids 1-247 of SEQ ID NO:4 (ACRP30) arecloned into mammalian expression vector pcDNA4HisMax. Polynucleotidesencoding a heterologous polypeptide comprised of human zinc-alpha2-glycoprotein signal peptide fused N-terminally to gOBG3 polypeptidefragment of the invention, wherein said gOBG3 polypeptide fragment isselected from amino acids 18-244, 19-244, 20-244, 21-244, 22-244,23-244, 24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244,32-244, 33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244,41-244, or 42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position36 is substituted by an amino acid other than cysteine; amino acids37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2; aminoacids 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247,35-247, 36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine atposition 39 is substituted by an amino acid other than cysteine; oramino acids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ IDNO:4, are cloned into mammalian expression vector pcDNA4HisMax.

Polynucleotides encoding polypeptides comprising amino acids 1-244 ofSEQ ID NO:2 (APM1) or amino acids 1-247 of SEQ ID NO:4 (ACRP30) arecloned into mammalian expression vector pcDNA3.1 Hygro. Polynucleotidesencoding a heterologous polypeptide comprised of human zinc-alpha2-glycoprotein signal peptide fused N-terminally to gOBG3 polypeptidefragment of the invention, wherein said gOBG3 polypeptide fragment isselected from amino acids 18-244, 19-244, 20-244, 21-244, 22-244,23-244, 24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244,32-244, 33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244,41-244, or 42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position36 is substituted by an amino acid other than cysteine; amino acids37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2; aminoacids 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247,35-247, 36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine atposition 39 is substituted by an amino acid other than cysteine; oramino acids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ IDNO:4, are cloned into mammalian expression vector pcDNA3.1Hygro.

Alternatively, polynucleotides encoding for polypeptides comprisingamino acids 1-244, 18-244, 19-244, 20-244, 21-244, 22-244, 23-244,24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244,33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244, 41-244,or 42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position 36 issubstituted by an amino acid other than cysteine; amino acids 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ D NO:2; amino acids1-247, 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247,35-247, 36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine atposition 39 is substituted by an amino acid other than cysteine; oramino acids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ IDNO:4 are cloned into any bacterial, mammalian, or baculoviral expressionvector, preferably selected from pTrcHis, pET30a, FastBacHT, pcDNA4His,or pcDNA3. Hygro. Further alternatively, polynucleotides encoding aheterologous polypeptide comprised of human zinc-alpha 2-glycoproteinsignal peptide fused N-terminally to gOBG3 polypeptide fragment of theinvention, wherein said gOBG3 polypeptide fragment is selected fromamino acids 18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244,25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244,34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position 36 issubstituted by an amino acid other than cysteine; amino acids 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2; amino acids18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247, 26-247,27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247, 35-247,36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247, 44-247,or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine at position 39 issubstituted by an amino acid other than cysteine; or amino acids 40-247,41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4, are clonedinto any bacterial, mammalian, or baculoviral expression vector,preferably selected from pTrcHis, pET30a, FastBacHT, pcDNA4His, orpcDNA3. Hygro.

Alternatively, polynucleotides encoding for polypeptides selected fromamino acids 1-244, 18-244, 19-244, 20-244, 21-244, 22-244, 23-244,24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244, 32-244,33-244, 34-244, 35-244, 36-244, 37-244, 38-244, 39-244, 40-244, 41 -244,or 42-244 of SEQ ID NO:2 (APM1) wherein the cysteine at position 36 issubstituted by an amino acid other than cysteine; amino acids 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2; amino acids1-247, 18-247, 19-247, 20-247, 21-247, 22-247, 23-247, 24-247, 25-247,26-247, 27-247, 28-247, 29-247, 30-247, 31-247, 32-247, 33-247, 34-247,35-247, 36-247, 37-247, 38-247, 39-247, 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4 (ACRP30) wherein the cysteine atposition 39 is substituted by an amino acid other than cysteine; oramino acids 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ IDNO:4 are cloned into Pichia Pastoris (yeast) expression vector,preferably PHIL-S1. Further alternatively, polynucleotides encoding aheterologous polypeptide comprised of human zinc-alpha 2-glycoproteinsignal peptide fused N-terminally to gOBG3 polypeptide fragment of theinvention, wherein said gOBG3 polypeptide fragment is selected from18-244, 19-244, 20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244,27-244, 28-244, 29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244,36-244, 37-244, 38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2(APM1) wherein the cysteine at position 36 is substituted by an aminoacid other than cysteine; amino acids 37-244, 38-244, 39-244, 40-244,41-244, or 42-244 of SEQ ID NO:2; amino acids 18-247, 19-247, 20-247,21-247, 22-247, 23-247, 24-247, 25-247, 26-247, 27-247, 28-247, 29-247,30-247, 31-247, 32-247, 33-247, 34-247, 35-247, 36-247, 37-247, 38-247,39-247, 40-247, 41-247, 42-247, 43-247, 44-247, or 45-247 of SEQ ID NO:4(ACRP30) wherein the cysteine at position 39 is substituted by an aminoacid other than cysteine; or amino acids 40-247, 41-247, 42-247, 43-247,44-247, or 45-247 of SEQ ID NO:4, are cloned into Pichia Pastoris(yeast) expression vector, preferably PHIL-S1.

Cell growth and induction of OBG3 expression: DH5alpha E. coli hostcells were transformed with a pTrcHis expression vector containingpolynucleotides of SEQ ID NO:1 encoding for a polypeptide comprisingamino acid residues 110-244 of APM1 (SEQ ID NO:2), also referred to asgAPM1 (110-244). Cells were grown overnight at room temperature (˜22 °C.) and induced at an approximate density of 0.45 OD₆₀₀. The cells wereinduced with 1 mM IPTG for 6 hours at room temperature. Cells wereharvested and lysed by French Press and sonication in buffer comprising50 mM Bis-Tris-Propane, pH 8.75, 1% Triton X-100, 5 mM MgCl₂ and 5 mMCaCl₂ and 50 mM NaCl. Extracts were centrifuged to obtain a solublefraction. The soluble protein in the supernatant fraction was passedover a Ni-affinity column at 5° C. The column was then washed with 25 mMimidazole. Alternatively, several washes of increasing imidazole up to25 mM is used. The gAPM1 (110-224) protein was eluted from the columnwith a concentration of 100 mM imidazole. The eluant comprising thegAPM1 (110-244) fraction was run over an ion exchange column using DEAEresin. Alternatively, a hydroxyapatite column is used. The gAPM1(110-244) protein was eluted with high salt buffer and dialyzed toreduce the salt concentration. This material was used in Example 18 toassay affect on FFA oxidation.

Example 17 Effect of gAPM1 on Free Fatty Acid Oxidation

Bacterially expressed gAPM1-His tag (110-244), gACRP30-his tag (104-247)and gACRP30 (104-247)-N-term-His/C-term-Flag tagged proteins werecompared for activity in a free fatty acid (FFA) oxidation assay.Briefly, C2C12 cells and SKMC cells were grown to ˜95% confluency. Cellswere differentiated for 7 days. Cells were starved overnight in serumfree DME containing 0.5% Albumax I (fatty acid rich BSA). Cells werethen preincubated in medium comprising Basal Medium Eagle (BME), 20 mMHEPES, pH7.4, 4 mM L-Glutamine, 1% fatty acid free BSA (0.15 nM). (As anegative control, a flask with no cells was included with 2 ml ofPreincubation media alone.) Cells were treated with either 5.0 ug/mlgACRP30-trypsin-cleaved (0.275 mg/ml), 5.0 ug/ml gACRP30 bacteriallyexpressed (0.36 mg/ml), 5.0 ug/ml gACRP30 His-Flag tagged bacteriallyexpressed (0.24 mg/ml), 5.0 ug/ml gAPM1 (110-244) (0.09 mg/ml)bacterially expressed (bacterial expression described in Example 17) for2 hours at 37° C. As a positive control, 100 uM isoproteranol was addedto the cells 15 minutes before addition of perchloric acid.

Treated cells were assayed as follows:

-   -   1. 100 ul of media with 4 mM palmitic acid and 4 uCi/ml        14C-palmitic acid was added to each of the flasks, and cells        were incubated at 37° C. for 90 minutes.    -   2. Using a 1 ml Luer-lok syringe, Solvable was injected into the        center well containing a filter paper approximately 15 min        before adding the perchloric acid.    -   3. Using a 1 ml Luer-lok syringe, 1 ml perchloric acid was added        and cells were incubated for 1 hour at 37° C.    -   4. After 1 hour, 15 ml Hionic Fluor was added and filter paper        was transferred into appropriate scintillation vial and shaken        for 30 min at 4° C., then counted.

The N-terminally His tagged gAPM1 (110-244), gACRP30 (104-247) andisoproterenol (positive control) significantly increased FFA oxidationabove non-treated cells. The recombinant mouse protein containing bothan N-term His tag and a C-term FLAG tag was not active in this assay.

Example 18 In Vivo Evaluation of OBG3 of gOBG3 Polypeptide Fragments ofthe Invention

7-Day Evaluation: A study protocol was established to evaluate theeffects on lipid or glucose metabolism of isolated and purifiedrecombinant gOBG3 polypeptides of the invention, for example, aminoacids 101-244, 105-244, 110-244, 19-244, 111-191, 191-244, and 144-199of SEQ ID NO:2, and amino acids 104-247 of SEQ ID NO:4, and His-taggedanalogues thereof. This protocol can be expanded to include theefficient screening of any polypeptide for potential use in methods toalter lipid or glucose metabolism. A feature of this protocol is theshort-term experimental length as well as detection of intermediate-termexperimental effects. Mice are treated for a total of 7 days. At thestart of the treatment acute effects either on fat/glucose metabolism oron hepatic glucose output are measured by conducting a postprandiallipemia study (PPL) or an Insulin tolerance test (ITT), respectively.Short-term effects on blood chemistry are determined after 3-4 days oftreatment and a complete analysis is performed at the end of thetreatment period. This includes a second study, either PPL, ITT or OGTT(oral glucose tolerance test). In addition effects on body weight, bodycomposition (% lean and fat tissue), fat storage in liver and muscle aremeasured and tissues are isolated to follow effects on gene expressionor to determine histological changes according to routine methods knownin the art.

I. Assay for Glucose Tolerance

A standard Glucose Tolerance Test (GTT) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted for 3-6 hours. The standard model uses        C57B1/6 male mice kept on a high fat diet, this results in        decreased insulin sensitivity.    -   2. A baseline blood sample is taken from the tail and the        glucose level is measured using glucose test strips and the ‘One        Touch Ultra System’ (Johnson & Johnson), as with all following        blood draws.    -   3. Treatment with test polypeptide (5-150 ug/50 ul/mouse) is        given by i.p. injection. Control animals are injected with equal        volume of saline.    -   4. Immediately following treatment, glucose is injected i.p.        (Dose: 1 g/kg body weight).    -   5. After all injections are done, the plasma glucose level is        monitored at 30′, 60′, 90′, and 120′ post-treatment.    -   6. For the 7-day evaluation, mice are injected at the same time        everyday for 7 consecutive days, and GTT assay is performed on        each day according to step 5 above.        II. Assay for Insulin Tolerance

A standard Insulin Tolerance Test (ITT) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted for 3-6 hours. The standard protocol uses        C57B1/6 male mice kept on a high fat diet, this results in        decreased insulin sensitivity.    -   2. A baseline blood sample is taken from the tail and the        glucose level is measured using glucose test strips and the ‘One        Touch Ultra System’ (Johnson & Johnson), as with all following        blood draws.    -   3. Treatment with test protein (5-150 ug/50 ul/mouse) is given        by i.p. injection, Untreated animals are injected with equal        volume of saline.    -   4. Immediately following treatment insulin is injected (Dose:        0.31 U/kg body weight).    -   5. The plasma glucose level is monitored at 15′, 30′, 60′, 90′,        and 120′ post-treatment.    -   6. For the 7-day evaluation, mice are injected at the same time        everyday for 7 consecutive days, and ITT assay is performed on        each day according to step 5 above.        III. Assay for Postprandial Lipemia

A standard Postprandial lipemia assay (PPL) following injection of testpolypeptides is performed on mice as follows:

-   -   1. Mice are fasted for 3-6 hours. The standard protocol uses        normal C57B1/6 male mice.    -   2. A baseline blood sample is taken from the tail.    -   3. A high fat/high sucrose test meal (6 g butter, 6 g sunflower        oil, 10 g non fat dry milk, 10 g sucrose, 12 ml distilled water        prepared fresh) is given by gavage (Dose: 1% of the animal total        body weight in ml).    -   4. Following the test meal, animals are treated with test        protein (5-150 ug/50 ul/mouse) given by i.p. injection.        Untreated animals are injected with equal volume of saline. This        treatment is repeated each day throughout a 7-day evaluation        study.    -   5. Blood samples are taken at 1, 2, 3 and 4 hrs following        treatment. Samples are immediately stored on ice until plasma        separation.    -   6. Plasma samples are assayed for metabolic parameters including        glucose, insulin, leptin, triglycerides, and FFAs.

Plasma glucose concentration is determined as described above in the GTTassay using the ‘One Touch Ultra System’. Alternatively, a standardglucose test kit is used (Sigma). The concentration of triglycerides andfree fatty acids (FFAs) are determined using calorimetric assays(Triglycerides, Sigma; FFA, Wako Biochemicals, Osaka). Insulin andLeptin are determined by RIA (Linco Research, St Charles, Mo.).

In addition to the 7-day evaluation study protocol, GTT, ITT, and PPLassays can be performed in any length study, including single day,short-term, and long-term periods following injection of the testpolypeptides.

Example 19 Infant Formula Supplementation

The following example describes a method of administering gOBG3polypeptides to newborns as supplemental nutritional support and furtherprovides a method of promoting growth of an infant by administeringgOBG3-fortified human breast milk or gOBG3-fortified breast milksubstitute formulation from a nonhuman source. Dehydrated or lyophilizedgOBG3 polypeptide powder is directly added to pumped human breast milk(freshly pumped or prewarmed after storage) or any prewarmed breast milksubstitute formulation from a nonhuman source, in a range of 5-1000ng/ml, preferably 20-800 ng/ml, more preferably 65-650 ng/ml.Supplementation with polypeptides of the invention is provided toinfants, particularly pretern infants, in bottle feedings of humanbreast milk or breast milk substitute at every feeding throughout theday, and is continued to be provided from birth to 6 months of age.Preterm infants may be of low birth weight or very low birth weight.

A primary objective of the study is to demonstrate that a polypeptide ofthe invention added to human milk (HM) or human milk substitute (HMS)supports acceptable growth in preterm infants. A second objective is toevaluate the serum biochemistries (ie, protein status, calcium, alkalinephosphatase), tolerance, clinical problems, and morbidity of prematureinfants consuming the nutritional module. Another secondary objective isto compare the supplemental composition of the instant invention to acommercial fortifier powder that has been in use for a number of yearsto promote growth in preterm infants. An intent-to-treat, prospective,randomized, double-blinded multicenter study is conducted to evaluatepreterm infants receiving preterm milk supplemented with either acommercially available powdered human milk fortifier (Enfamil.RTM. HumanMilk Fortifier, control) or the supplemental polypeptide (test) powderof the current invention (experimental) at every feeding. Subjects areenrolled and randomized to each fortifier powder prior to 21 days oflife. Study Day 1 is when fortification of the test powder begins andthe subject reaches an intake of at least 100 mL/kg/day. Anthropometricindices, serum biochemistries, intake, tolerance, and morbidity data areassessed. Each infant is studied until hospital discharge; onlyanthropometric variables (weight, length, and head circumference) arecollected after Study Day 29. Premature infants are recruited fromneonatal intensive care units that had agreed to collaborate with studyinvestigators. Single, twin, or triplet infants born around 33 weeksgestational age, with appropriate weight for gestational age, andweighing around 1600 g are eligible to participate. One-hundred andforty-four infants are randomized to either control or experimental; 70preterm infants are randomized to the control group and 74 preterminfants are randomized to the experimental group. The randomization isproportional for birth weight and gender.

The independent variables (treatments) are the control fortifier powderand the experimental test powder which are added to HM or HMS. Bothfortifiers (test and control) are provided in small packets in powderedform and are added to 25 mL HM or HMS. The primary outcome variable isweight gain (g/kg/day) from study day 1 to study day 29 or discharge,whichever comes first. Secondary outcome variables are length gain(mm/day) and serum biochemistries to evaluate protein status,electrolyte status, mineral homeostasis, and vitamin A and E status.Serum biochemistries also include unscheduled laboratory results to berecorded in the medical chart. Tertiary variables include headcircumference gain (mm/day), clinical history, intake, tolerance,clinical problems/morbidity, respiratory status, antibiotic use, and thenumber of transfusions. Mean total energy intakes during the studyperiod is not different between the groups, around 118 kcal/kg/day.

Example 20 Assessment of Homotrimer Formation by gOBG3 PolypeptideFragment

Homotrimer formation by gOBG3 polypeptide fragment is assessed usingsedimentation equilibrium in analytical centrifuges, a method thatdetermines molecular weight accurately and independently of otherphysical factors such as shape.

Candidate gOBG3 polypeptide fragment homotrimer is purified, for exampleusing a protocol comprising a method of gel filtration such as 16/60superdex 200 gel filtration column (Amersham). Said purified candidategOBG3 polypeptide fragment homotrimer protein concentration is made 3 μMin 5.7 mM phosphate (pH 7.5), 137 mM NaCl, 2.7 mM KCl. Samples arecentrifuged at 8,000 rpm for 18 hours at 10° C. in a Beckman XL-Aanalytical ultracentrifuge before absorbance is recorded. The data arefit globally, using MacNonlin PPC to the following equation thatdescribes the sedimentation of a homogeneous species: Abs=B+A′ exp[H×M(x²−x₀ ²] where Abs =absorbance at radius x, A′=absorbance at referenceradius x₀, H=(1−νρ)ω²/2RT, R=gas constant, T=temperature in Kelvin,ν=partial specific volume=0.71896131 mL/g, ρ=density of solvent=1.0061g/ml, ω=angular velocity in radians/s, M=apparent molecular weight, andB=solvent absorbance (blank).

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1. A method of lowering circulating free fatty acid levels in anindividual comprising administering to said individual a compositioncomprising a carrier and a homotrimeric gOBG3 polypeptide fragment,wherein said gOBG3 polypeptide fragment is selected from 18-244, 19-244,20-244, 21-244, 22-244, 23-244, 24-244, 25-244, 26-244, 27-244, 28-244,29-244, 30-244, 31-244, 32-244, 33-244, 34-244, 35-244, 36-244, 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2 wherein thecysteine at position 36 is substituted with serine.
 2. The method ofclaim 1, wherein said method further reduces body mass.
 3. An isolatedhomotrimeric gOBG3 polypeptide fragment, wherein said gOBG3 polypeptidefragment is selected from 18-244, 19-244, 20-244, 21-244, 22-244,23-244, 24-244, 25-244, 26-244, 27-244, 28-244, 29-244, 30-244, 31-244,32-244, 33-244, 35-244, 36-244. 37-244, 38-244, 35-244, 36-244, 37-244,38-244, 39-244, 40-244, 41-244, or 42-244 of SEQ ID NO:2 wherein thecysteine at position 36 is substituted with serine.
 4. A compositioncomprising a carrier and the gOBG3 polypeptide fragment of claim
 3. 5.An isolated polynucleotide, or complement thereof, encoding the gOBG3polypeptide fragment of claim
 3. 6. A composition comprising a carrierand an isolated polynucleotide according to claim
 5. 7. A vectorcomprising an isolated polynucleotide sequence encoding the gOBG3polypeptide fragment of claim
 3. 8. A composition comprising a carrierand a vector of claim
 7. 9. A transformed host cell comprising thevector according to claim 7.